Disabling of Receptor Activator of Nuclear Factor-κB (RANK) Receptor Complex by Novel Osteoprotegerin-like Peptidomimetics Restores Bone Loss in Vivo

Abstract

The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-B (RANK) and osteoprotegerin (OPG), are known to be regulators of development and activation of osteoclasts in bone remodeling. Sustained osteoclast activation that occurs through TRANCE-RANK causes osteopenic disorders such as osteoporosis and contributes to osteolytic metastases. Here, we report a rationally designed small molecule mimic of osteoprotegerin to inhibit osteoclast formation in vitro and limit bone loss in an animal model of osteoporosis. One of the mimetics, OP3-4, significantly inhibited osteoclast formation in vitro (IC 50 ‫؍‬ 10 M) and effectively inhibited total bone loss in ovariectomized mice at a dosage of 2 mg/kg/day. Unlike soluble OPG receptors, which preclude TRANCE binding to RANK, OP3-4 shows the ability to modulate RANK-TRANCE signaling pathways and alters the biological functions of the RANK-TRANCE receptor complex by facilitating a defective receptor complex. These features suggest that OPG-derived small molecules can be used as a probe to understand complex biological functions of RANK-TRANCE-OPG receptors and also can be used as a platform to develop more useful therapeutic agents for inflammation and bone disease. TRANCE,1 a TNF family member, also known as OPGL, RANKL, ODF, and OCIF, plays a key role in the development of osteoclasts and in modulating their bone resorbing activity (1-4). Increased osteoclast activity has been reported in many osteopenic disorders, including postmenopausal osteoporosis, Paget's disease, bone metastases, and rheumatoid arthritis (5-7). TRANCE interacts with two receptors: a secreted decoy receptor osteoprotegerin, OPG (8), and a transmembrane receptor, RANK (9 -11). The interaction between TRANCE and RANK is essential for osteoclastogenesis because RANK is activated by TRANCE and then associates with TNF receptorassociated family members to trigger downstream signaling (11) events. OPG, on the other hand, plays an opposite role by preventing TRANCE from binding and activating RANK and thus is considered a "decoy" receptor. In this way, the interaction between TRANCE and OPG or TRANCE and RANK represents a complex network required for normal bone development, and any imbalance in the system potentially leads to bone disorders. Thus it is well recognized that this receptor complex network is an interesting drug development target for the treatment of bone disorders such as osteoporosis (12), and there is considerable interest in developing ways to modulate RANK functions which may prove beneficial for bone-related pathologies.To date there is no three-dimensional structural information available for the RANK receptor complex. However, it is believed that the complex should resemble that of the TNF receptor as a member of TNF superfamily. The TNF-␤⅐TNFR1 co-crystal structure has been solved, thus facilitating rational drug design based on the receptor-ligand interaction sites coupled wi...

Structure-based development of a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor peptide and molecular basis for osteopetrosis

“…Consequently, RANK-Fc, Fc-OPG, and anti-RANKL antibodies have been developed as therapeutics for osteoporosis (13)(14)(15)(16)(17)(18)(19). Alternatively, peptide mimics of OPG (OP3-4 peptide) (20,21) and the tumor necrosis factor (TNF) receptor (WP9QY peptide) (22) were also developed and showed inhibitory activity against the RANKL-induced osteoclastogenesis.…”

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

“…Given that OP3‐4 binds to RANKL to a similar extent as that to W9 17, 18, these data suggest that both W9 and OP3‐4 stimulate osteoblast differentiation through membrane‐bound RANKL in osteoblasts. Additional experiments showing that a mutation in the intracellular domain of RANKL reduces the phosphorylation of Akt and S6K1 would therefore be necessary to confirm the existence of the RANKL‐binding peptide‐induced RANKL‐reverse signals.…”

“…S2), although W9 and OP3‐4 are reported to inhibit the osteoclast differentiation induced by RANKL 17, 18. Considering the life span of murine osteoclasts, which is generally believed to be around 3–4 days in vivo, these data on osteoclasts suggest that the peptide‐release from the gelatin hydrogel carrier did not take place in the last phase of the experiment.…”

“…The other cyclic peptide W9 (YCWSQYLCY) and the control peptide (FCYISEVEDECY) 17, 26 were purchased from the American Peptide Company (Sunnyvale, CA, USA). Recombinant human BMP‐2 was provided by Yamanouchi Pharmaceutical Co., Ltd. (Current Astellas Phrma Inc.; the patent has since been transferred to Bioventus LLC [Durham, NC, USA]).…”

“…OP3‐4, another peptide, which was recently demonstrated to be a systemic stimulator of bone formation in an inflammatory bone destruction model 15, was originally designed to mimic osteoprotegerin, a natural antagonist of receptor activator of NF‐κB ligand (RANKL) 17. Both W9 and OP3‐4 have been shown to bind to RANKL, a stimulator of osteoclastogenesis, thereby inhibiting osteoclast formation and bone resorption 17, 18. However, the reason why these RANKL‐binding peptides stimulate bone formation remains to be clarified 19, 20.…”