FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073112480> ?p ?o ?g. }

• W2073112480 endingPage "8277" @default.
• W2073112480 startingPage "8269" @default.
• W2073112480 abstract "The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-κB (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 (IC50 = 10 μm) 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. The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-κB (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 (IC50 = 10 μm) 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, 1The abbreviations used are: TRANCE, tumor necrosis factor-related activation-induced cytokine; CSF, colony-stimulating factor; ERK, extracellular signal-regulated kinase; HPLC, high performance liquid chromatography; M-CSF, macrophage-colony-stimulating factor; NF-κB, nuclear factor-κB; OPG, osteoprotegerin; OVX, ovariectomized; pQCT, peripheral quantitative computed tomography; RANK, receptor activator of nuclear factor-κB; TNF, tumor necrosis factor; TNFR, TNF receptor; TRAP, tartrate-resistant acid phosphatase. 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 (1Nakagawa N. Kinosaki M. Yamaguchi K. Shima N. Yasuda H. Yano K. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 253: 395-400Crossref PubMed Scopus (622) Google Scholar, 2Michigami T. Ihara-Watanabe M. Yamazaki M. Ozono K. Cancer Res. 2001; 61: 1637-1644PubMed Google Scholar, 3Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3018) Google Scholar, 4Theill L.E. Boyle W.J. Penninger J.M. Annu. Rev. Immunol. 2002; 20: 795-823Crossref PubMed Scopus (673) Google Scholar). Increased osteoclast activity has been reported in many osteopenic disorders, including postmenopausal osteoporosis, Paget's disease, bone metastases, and rheumatoid arthritis (5Jones D.H. Kong Y.Y. Penninger J.M. Ann. Rheum. Dis. 2002; 61: 32-39Crossref PubMed Scopus (114) Google Scholar, 6Mori H. Kitazawa R. Mizuki S. Nose M. Maeda S. Kitazawa S. Histochem. Cell Biol. 2002; 117: 283-292Crossref PubMed Scopus (51) Google Scholar, 7Wang R. Zhang L. Zhang X. Moreno J. Celluzzi C. Tondravi M. Shi Y. Eur. J. Immunol. 2002; 32: 1090-1098Crossref PubMed Scopus (62) Google Scholar). TRANCE interacts with two receptors: a secreted decoy receptor osteoprotegerin, OPG (8Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T.M. Hill D. Pattison W. Campbell P. Boyle W.J. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4350) Google Scholar), and a transmembrane receptor, RANK (9Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar, 10Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 11Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1419) Google Scholar). The interaction between TRANCE and RANK is essential for osteoclastogenesis because RANK is activated by TRANCE and then associates with TNF receptor-associated family members to trigger downstream signaling (11Hsu H. Lacey D.L. Dunstan C.R. Solovyev I. Colombero A. Timms E. Tan H.L. Elliott G. Kelley M.J. Sarosi I. Wang L. Xia X.Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M.B. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1419) Google Scholar) 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 (12Khosla S. Endocrinology. 2001; 142: 5050-5055Crossref PubMed Scopus (923) Google Scholar), 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-β·TNFR1 co-crystal structure has been solved, thus facilitating rational drug design based on the receptor-ligand interaction sites coupled with rational ligand-receptor mutation studies (13Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (984) Google Scholar). The crystal structure of the TNF receptor both in complexed and uncomplexed forms provides a general structural framework for understanding the atomic surfaces these receptors use to bind to their ligands (14Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar, 15Eck M.J. Ultsch M. Rinderknecht E. de Vos A.M. Sprang S.R. J. Biol. Chem. 1992; 267: 2119-2122Abstract Full Text PDF PubMed Google Scholar, 16Naismith J.H. Sprang S.R. J. Inflamm. 1995; 47: 1-7PubMed Google Scholar). We have proposed that the modular organization of structures of biological proteins and the identification of localized ligand-receptor interaction sites permit the rational design of smaller, peptidomimetics that can interfere with these ligand-receptor interactions (17Idriss H.T. Naismith J.H. Microsc. Res. Tech. 2000; 50: 184-195Crossref PubMed Scopus (662) Google Scholar). The mimetics on their own may be therapeutically active but also can be used to guide authentic pharmaceutical designs. We have shown that a small peptide sequences derived from the TNFR1 surface which interacts with the ligand has reasonable activity in blocking the biological actions of the TNF receptor (18Takasaki W. Kajino Y. Kajino K. Murali R. Greene M.I. Nat. Biotechnol. 1997; 15: 1266-1270Crossref PubMed Scopus (118) Google Scholar, 19Murali R. Greene M.I. Immunol. Res. 1998; 17: 163-169Crossref PubMed Scopus (27) Google Scholar). In this study, we have identified several critical binding sites on TRANCE and OPG based on a three-dimensional structural model of the OPG-TRANCE receptor complex. The models were developed on the basis of co-crystal structures of TNF-β·TNFR1 (13Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (984) Google Scholar) and the deduced crystal structure of TRANCE (20Ito S. Wakabayashi K. Ubukata O. Hayashi S. Okada F. Hata T. J. Biol. Chem. 2002; 277: 6631-6636Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Lam J. Nelson C.A. Ross F.P. Teitelbaum S.L. Fremont D.H. J. Clin. Invest. 2001; 108: 971-979Crossref PubMed Scopus (157) Google Scholar). The putative critical contacts were then used for the design of exocyclic peptidomimetics. In general the small molecules designed from OPG appear to inhibit TRANCE binding to RANK more effectively than mimetics designed from TRANCE. Materials and Cell Cultures—A soluble form of recombinant TRANCE has been described previously (22Josien R. Li H.L. Ingulli E. Sarma S. Wong B.R. Vologodskaia M. Steinman R.M. Choi Y. J. Exp. Med. 2000; 191: 495-502Crossref PubMed Scopus (286) Google Scholar). Recombinant human M-CSF, RANK, and OPG were obtained from PeproTECH (Rocky Hill, NJ). All other chemicals were from Sigma. Cell Lines and Culture Systems—RAW264.7 cells were from American Type Culture Collection (Manassas, VA) and were cultured in RPMI (Invitrogen) containing 10% fetal bovine serum. Murine osteoclast precursors were from 6-8-week-old C57BL/6 male mice (Jackson Laboratory, Bar Harbor, ME). Murine bone marrow cells were cultured in α-minimal essential medium (Invitrogen) containing 10% fetal bovine serum and 5 ng/ml M-CSF for 12 h in 100-mm-diameter dishes at a density of 1 × 107/dish. The nonadherent cells were then harvested and cultured with 30 ng/ml M-CSF in 100-mm dishes at the same density as before for an additional 48 h. Floating cells were removed, and attached cells were used as osteoclast precursors for generating osteoclasts (23Takami M. Kim N. Rho J. Choi Y. J. Immunol. 2002; 169: 1516-1523Crossref PubMed Scopus (201) Google Scholar). Design of TRANCE and OPG Mimetics—Computer modeling was built using Modeler 4.0 (24Sali A. Blundell T.L. J. Mol. Biol. 1990; 212: 403-428Crossref PubMed Scopus (481) Google Scholar), and further refinement and analysis were performed using INSIGHT II (Molecular Simulation, Inc., San Diego). The OPG-TRANCE structure complex was initially modeled by molecular replacement using models constructed from crystallographic coordinates, Protein Data Bank (PDB) (25Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.E. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8178) Google Scholar). The structure model of OPG was built using substructures of TNFR (1TNR), Fas (1BZI), and TRAIL (1D4V and 1D0G) receptors. Models were checked for consistency, and the ambiguous loop regions were reexamined and built using CONGEN (26Bruccoleri R.E. Haber E. Novotny J. Nature. 1988; 335: 564-568Crossref PubMed Scopus (173) Google Scholar). In the final consensus model the side chains involved in unfavorable interactions were adjusted manually as a side chain conformation search was carried out. The model was then optimized using energy minimization and molecular simulation calculations. The accuracy of the model was checked using Ramachandran plot (27Ramachandran G.N. Venkatachalam C.M. Biopolymers. 1968; 6: 1255-1262Crossref PubMed Scopus (60) Google Scholar) and by profile analyses (28Zhang K.Y. Eisenberg D. Protein Sci. 1994; 3: 687-695Crossref PubMed Scopus (43) Google Scholar). The binding sites involved in direct interaction between TRANCE and OPG were analyzed and used as the initial template for peptide design, as shown in Table I. The model peptides designed were constructed from their sequences and folded using CHARMM. The folded peptides were minimized to convergence with a dielectric constants set to 80. Details of the design of receptor-based peptidomimetics have been described previously (18Takasaki W. Kajino Y. Kajino K. Murali R. Greene M.I. Nat. Biotechnol. 1997; 15: 1266-1270Crossref PubMed Scopus (118) Google Scholar, 29Murali R. Kajino K. Hasegawa A. Berezov A. Takasaki W. Ciliberto G. Savino R. Cytokine Inhibitors. Marcel Dekker, Inc., New York2000: 133-162Google Scholar).Table ISequence of critical TRANCE/OPG interaction sites and exocyclic peptidomimetics derived from the receptorDerived from binding sitesExocyclic peptidomimeticBinding site-1TRANCE74 YVTKTSIKIPSSH 86OL1-1YC VTKTSIKIPSSH CYOL1-2YC KTSIKIPSSH CYOPG70 YTDSWHTSD 78OP1-1YC PDSWH CY DEBinding site-2TRANCE61 SGDLATEY 68OL2-1YC GDLAT CYOL2-2YC SDFATE CYOPG82 YCSPVCKELQYVKQE 96OP2-1YC SKEL CY VKQEBinding site-3TRANCE96 YWSGNSEF 103OL3-1YC YWSNSEF CYOL3-2YC YW NSE CYOPG113 LEIEFCLKHR 122OP3-1YC EIEF CY KHROP3-2YC EIEF CYOP3-4YC EIEF CY LIR Open table in a new tab Peptide Synthesis and Cyclization—Linear peptides were ordered from the Protein Chemistry Laboratory, University of Pennsylvania. The purity and identity of peptides were confirmed by reverse-phase high performance liquid chromatography (HPLC) and mass spectrometry. The peptides were cyclized by air oxidation in distilled water adjusted to pH 8.0-8.5 with (NH4)2CO3 at 0.1 mg/ml, as described previously (18Takasaki W. Kajino Y. Kajino K. Murali R. Greene M.I. Nat. Biotechnol. 1997; 15: 1266-1270Crossref PubMed Scopus (118) Google Scholar). Kinetic Binding Studies by Surface Plasmon Resonance—Binding experiments were carried out on BiaCORE 3000 (BiaCORE, Uppsala, Sweden) instruments at 25 °C. Recombinant TRANCE, RANK, and OPG (PeproTECH, Inc.) were immobilized to the dextran hydrogel on the sensor surface (BiaCORE CM5 sensor chip) with a surface density of 2,000 resonance units. The surface was regenerated to remove all bound analyte among binding cycles using 0.2% SDS. The apparent rate constants (kon and koff) and the equilibrium binding constant (KD) for the receptor-peptide/ligand-peptide binding interaction were estimated from the kinetic analysis of sensorgrams, using the BIA evaluation 3.0 software (BiaCORE). Immunoblotting—To evaluate the effect of the peptide on the regulation of downstream molecules of TRANCE signaling during osteoclastogenesis, RAW264.7 cells (5 × 105/well) were cultured in 6-well plates for 12 h, treated with or without the designed peptide for 2 h, and then stimulated with TRANCE at 300 ng/ml for the indicated periods. Cells were then washed with ice-cold phosphate-buffered saline and lysed with lysis buffer. Cell lysates (15-30 μg) were separated by 12% SDS-PAGE, electroblotted onto nitrocellulose membranes (Osmonics, Westborough, MA), and probed with anti-phospho-IκBα, anti-IκBα, and anti-ERK2 antibodies (Cell Signaling Technology, Inc., Beverly, MA). The membranes were then developed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Effect on TRANCE-induced Osteoclast Formation and Pit Formation in Vitro—To evaluate the effect of the peptide on TRANCE-mediated osteoclastogenesis, murine osteoclast precursors were cultured in 96-well plates (1 × 105/ml, 200 μl/well) for 3 days in the presence of 30 ng/ml murine CSF-1 and 300 ng/ml sTRANCE with or without treatment of designed peptides at the indicated concentration. After 3 days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) as described previously (30Shiotani A. Takami M. Itoh K. Shibasaki Y. Sasaki T. Anat. Rec. 2002; 268: 137-146Crossref PubMed Scopus (63) Google Scholar). TRAP-positive multinucleated cells were counted as osteoclast-like multinucleated cells. For the pit formation assay, mouse osteoclast precursors (5 × 104 cells/0.2 ml/well) were placed on dentin slices (4 mm in diameter) in 96-well culture plates and cultured for 1 h with 30 ng/ml M-CSF. Dentine slices were then transferred into 48-well culture plates (Corning Glass). Cells on dentin slices were cultured in the presence of 30 ng/ml M-CSF with or without 200 ng/ml TRANCE for 4 days, in the absence or presence of peptide OP3-4 at the indicated concentration. Medium was replaced on day 3. On day 4, cells were removed from the dentin slices with cotton, and the slices were then immersed in Mayer's hematoxylin (Sigma) to stain the resorption pits formed by osteoclasts. Effect on Bone Resorption in Ovariectomized (OVX) Mice—In 8-week-old female C57BL/6 mice, the dorsal skin was incised, and ovaries were excised under anesthesia. The sham operation group and ovariectomy group were treated with peptide or vehicle only. An Arzet® osmotic pump (DURECT Co., Cupertino, CA) was used to deliver OP3-4 subcutaneously after the ovariectomy. The pump was used for systemic administration at a consistent rate for up to 4 weeks without repeated dosing. The pump was filled up to 200 μl for the duration (4 weeks) and delivered at the rate of 0.25 μl/h. The OVX mice were given peptide OP3-4 subcutaneously at a daily dosage of 2 mg/kg/day for 28 days. The mice were euthanized, and the tibia and femur were removed, cleaned of soft tissue, and fixed in 10% formalin. Femur were scanned by peripheral quantitative computed tomography (pQCT) to examine the total bone density. Animals were maintained in accordance with guidelines of Institutional Animal Care and Use Committee of the University of Pennsylvania. Molecular Modeling of TRANCE Binding to OPG and Development of OPG-like Mimetics—The overall topology of OPG is similar to that of the TNFR (Fig. 1A), although the homology in the primary structure is less than 50%. The three-dimensional model of OPG was built using modular fold of the TNFR superfamily (16Naismith J.H. Sprang S.R. J. Inflamm. 1995; 47: 1-7PubMed Google Scholar, 31Naismith J.H. Sprang S.R. Trends Biochem. Sci. 1998; 23: 74-79Abstract Full Text PDF PubMed Scopus (188) Google Scholar). The three-dimensional structure of OPG was built using substructures of TNFR (1TNR), Fas (1BZI), and TRAIL (1D4V and 1D0G) receptors. The structure of OPG consists of B2-A1-B2-A1-B1-B1-A1-B1 modular folds (16Naismith J.H. Sprang S.R. J. Inflamm. 1995; 47: 1-7PubMed Google Scholar, 31Naismith J.H. Sprang S.R. Trends Biochem. Sci. 1998; 23: 74-79Abstract Full Text PDF PubMed Scopus (188) Google Scholar, 32Bodmer J.L. Schneider P. Tschopp J. Trends Biochem. Sci. 2002; 27: 19-26Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). To study the fine details of the OPG-TRANCE complex, only the N-terminal region (22-185) was considered. The crystal structure of TRAIL-DR5 (33Cha S.S. Sung B.J. Kim Y.A. Song Y.L. Kim H.J. Kim S. Lee M.S. Oh B.H. J. Biol. Chem. 2000; 275: 31171-31177Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) was also used as template and used to obtain maximal receptor-ligand interaction. In the final analysis, the OPG-TRANCE complex was similar to the TRAIL-DR5 receptor complex (33Cha S.S. Sung B.J. Kim Y.A. Song Y.L. Kim H.J. Kim S. Lee M.S. Oh B.H. J. Biol. Chem. 2000; 275: 31171-31177Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Based on the structural analysis of TRANCE and OPG-TRANCE complex several contact sites have been identified in TRANCE and three sites on OPG (Fig. 1A). Sites on OPG were identified as following: TRANCE (Tyr74-His86) binds to OPG (Tyr70-Asp78); TRANCE (Ser61-Tyr68) binds to OPG(Tyr82-Glu96); and TRANCE (Tyr96-Phe103) binds to OPG (Leu113-Arg122). The sequences of the three binding sites from both ligand and receptor were then used as template for peptide design (Table I). Exocyclic peptidomimetics were created from the conformational features of the critical sites on TRANCE and OPG with the addition of aromatic residues. Cyclization of linear peptides was confirmed by 5,5′-dithiobis(nitrobenzoic acid). Purity was evaluated by reverse-phase HPLC before and after cyclization. All of the cyclized peptides were enriched to a purity of >98% before use. The purified exocyclic peptides were soluble and used to make a stock solution at 1 mm in phosphate-buffered saline (pH 7.4). Inhibition of sTRANCE-induced Osteoclast Formation in Vitro—We have designed several peptide mimetics from both OPG and TRANCE (Table I) and have screened them for biological activity. We have evaluated these mimetics at a 30 μm concentration on osteoclastogenesis in murine bone marrow cells co-cultured with TRANCE and CSF-1 (30Shiotani A. Takami M. Itoh K. Shibasaki Y. Sasaki T. Anat. Rec. 2002; 268: 137-146Crossref PubMed Scopus (63) Google Scholar). Several mimetics at a 30 μm concentration caused some reduction in the number of TRAP-positive multinucleated cells formed. Among all 11 peptides we designed from the three critical binding sites, 8 peptides exhibited 20-60% inhibitory activity (Fig. 1B). The peptides designed from OPG (OP series) showed a greater inhibitory effect than those designed from TRANCE (OL series). These data suggest that receptor-derived mimetics block ligand binding to its receptor differently than ligand mimetics. We have regarded the receptor as a better target in structure-based design and improved it further. The peptides derived from binding site 3, from both ligands (OL3-1, OL3-2) and receptor (OP3-1, OP3-2, OP3-4), exhibited more inhibitory effects compared with other derived peptides. This observation implied that binding site 3 may be an important binding surface for the signal competence of the OPG·TRANCE complex. Similar results were obtained when we designed peptides derived from the TNFR based upon the TNF-TNFR complex, indicating that membrane proximal domains may frequently be used as a critical interaction surface between ligands and receptors in the TNF-TNFR superfamily (18Takasaki W. Kajino Y. Kajino K. Murali R. Greene M.I. Nat. Biotechnol. 1997; 15: 1266-1270Crossref PubMed Scopus (118) Google Scholar). Earlier when we engineered a monoclonal antibody mimetic, AHNP, a HERCEPTIN mimetic (34Park B.W. Zhang H.T. Wu C. Berezov A. Zhang X. Dua R. Wang Q. Kao G. O'Rourke D.M. Greene M.I. Murali R. Nat. Biotechnol. 2000; 18: 194-198Crossref PubMed Scopus (167) Google Scholar), we found that residues in the exocyclic regions also play a critical role in stabilizing the conformation of amino acids within the cyclic ring. To see whether such an addition to OP3 would have any affect, we modified the sequence of OP3-1 by either removing the tail amino acid sequence of KHR as in the OP3-2 species or changing the amino acids KHR to LIR as in the OP3-4 forms. Subsequently we found that changing the residues in the exocyclic rings does affect the activity of the mimetic, indicating that the tail composed of KHR is critical for forming the binding site structure. The dissociation rate of mimetics has been shown to correlate with biological activity (35Jimi E. Akiyama S. Tsurukai T. Okahashi N. Kobayashi K. Udagawa N. Nishihara T. Takahashi N. Suda T. J. Immunol. 1999; 163: 434-442PubMed Google Scholar), and thus replacing KHR with LIR at the C terminus of OP3-4 actually enhanced the interaction between ligand and receptor, and consequently OP3-4 has become the most potent mimetic designed. Kinetic Binding Ability of OP3-4—Binding of OP3-4 to TRANCE was studied using surface plasmon resonance. OP3-4 bound to immobilized TRANCE in a dose-dependent manner (Fig. 2a). Based on the association and dissociation kinetics obtained using a 1:1 Langmuir model for simple bimolecular interactions, the binding affinity (Kd) of OP3-4 to TRANCE was 3.89 × 10-6m. The apparent association constant (kon) and disassociation constant (koff) rate constants were estimated to be 2.41 × 102m-1 s-1 and 9.37 × 10-4 s-1, respectively. The affinity of OP3-4 binding to TRANCE is 2 orders of magnitude less than the affinity TRANCE to RANK (Kd = 1.39 × 10-8m) or OPG (Kd = 1.76 × 10-8m), but the koff, which is a better indicator of biological activity (35Jimi E. Akiyama S. Tsurukai T. Okahashi N. Kobayashi K. Udagawa N. Nishihara T. Takahashi N. Suda T. J. Immunol. 1999; 163: 434-442PubMed Google Scholar), is comparable. (TRANCE to RANK koff = 3.05 × 10-4 s-1 and TRANCE to OPG koff = 2.4 × 10-4 s-1, respectively), The comparable koff suggests that the peptide can form a relatively stable complex with TRANCE and, as a consequence, compete in the interaction between ligand and receptor. To see further whether OP3-4 can block or compete with TRANCE binding to RANK, OP3-4 was studied in a competition assay. In a competition assay, OP3-4 reduced TRANCE binding to immobilized RANK in a dose-dependent manner. OP3-4 at a concentration of 50 μm significantly inhibited TRANCE binding to its receptor RANK (Fig. 2b). In Vitro Analysis of OP3-4 Mimetic—The inhibitory activity of peptide OP3-4 was then examined in terms of its ability to inhibit TRANCE-induced osteoclast formation (Fig. 3). Peptide concentrations of 2 μm or more caused a dose-dependent decrease in the number of TRAP-positive (TRAP+) multinucleated cells formed (Fig. 3A). In the presence of 50 μm OP3-4, the number of TRAP+ multinucleated cells was less than 10% of the number formed in co-cultures performed without the peptide. The IC50 was 10 μm. The decrease in the number of TRAP+ cells was apparently not the result of toxicity of the peptide because there was no change in the number of osteoblasts detected by staining for alkaline phosphatase (data not shown), nor was there a cytotoxic effect of 100 μm OP3-4 on RAW264.7 murine myeloid cells (data not shown). TRANCE stimulates bone resorption by mature osteoclasts (35Jimi E. Akiyama S. Tsurukai T. Okahashi N. Kobayashi K. Udagawa N. Nishihara T. Takahashi N. Suda T. J. Immunol. 1999; 163: 434-442PubMed Google Scholar, 36Burgess T.L. Qian Y. Kaufman S. Ring B.D. Van G. Capparelli C. Kelley M. Hsu H. Boyle W.J. Dunstan C.R. Hu S. Lacey D.L. J. Cell Biol. 1999; 145: 527-538Crossref PubMed Scopus (608) Google Scholar). We therefore examined the effect of OP3-4 on TRANCE-induced bone resorption in vitro (Fig. 3C). Mature osteoclasts from neonatal mice were plated on dentin slices. The cells were cultured for 4 days with 200 ng/ml TRANCE alone or with 30 μm OP3-4 and TRANCE. As reported previously (35Jimi E. Akiyama S. Tsurukai T. Okahashi N. Kobayashi K. Udagawa N. Nishihara T. Takahashi N. Suda T. J. Immunol. 1999; 163: 434-442PubMed Google Scholar, 36Burgess T.L. Qian Y. Kaufman S. Ring B.D. Van G. Capparelli C. Kelley M. Hsu H. Boyle W.J. Dunstan C.R. Hu S. Lacey D.L. J. Cell Biol. 1999; 145: 527-538Crossref PubMed Scopus (608) Google Scholar), 200 ng/ml TRANCE stimulated bone resorption by nearly 3-fold. OP3-4 significantly blocked the increased bone resorption at 30 μm. The number of osteoclasts on the dentin slices were not changed by TRANCE alone or by OP3-4 plus TRANCE. Thus, interfering with TRANCE binding to RANK by the use of designed peptidomimetics was effective in inhibiting TRANCE-induced osteoclast formation in vitro. Down-modulation of NF-κB by OP3-4—NF-κB activation in osteoclast precursors has been implicated as a signaling pathway involved in the successful differentiation of precursors to mature osteoclasts (10Darnay B.G. Haridas V. Ni J. Moore P.A. Aggarwal B.B. J. Biol. Chem. 1998; 273: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Activation of NF-κB is controlled by an inhibitory subunit, IκB, which retains NF-κB in the cytoplasm. NF-κB activation requires the sequential activation, phosphorylation, ubiquitination, and degradation of IκB as well as consequent exposure of discreet nuclear localization signals on the NF-κB surface (37Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2451) Google Scholar). As shown in Fig. 4, TRANCE markedly induced the phosphorylation of IκB-α by 5 min, and a concurrent degradation of IκBα. In Fig. 4, OP3-4 inhibited TRANCE-stimulated NF-κB activation as measured by lack of IκB-α phosphorylation and degradation. OP3-4 thus inhibited TRANCE-induced osteoclast formation at least in part through suppressing the NF-κB pathway. Although TRANCE also activates c-Jun N-terminal kinase (38Lee Z.H. Kwack K. Kim K.K. Lee S.H. Kim H.H. Mol. Pharmacol. 2000; 58: 1536-1545Crossref PubMed Scopus (59) Google Scholar), OP3-4 did not exhibit an inhibitory or stimulatory effect on this pathway (data not shown). OP3-4 Protects Mice against Ovariectomy-associated Bone Loss—It is well known that ovariectomy results in reduced bone mineral density associated with loss of estrogen (39Sato M. Zeng G.Q. Turner C.H. Endocrinology. 1997; 138: 4330-4337Crossref PubMed Scopus (124) Google Scholar). It was important to demonstrate that OP3-4 in vivo can protect against pathological decreases of bone density in OVX animals. The pQCT technique measu" @default.
• W2073112480 created "2016-06-24" @default.
• W2073112480 creator A5001874444 @default.
• W2073112480 creator A5014959620 @default.
• W2073112480 creator A5017857303 @default.
• W2073112480 creator A5054190325 @default.
• W2073112480 creator A5068837376 @default.
• W2073112480 creator A5079411311 @default.
• W2073112480 date "2004-02-01" @default.
• W2073112480 modified "2023-10-10" @default.
• W2073112480 title "Disabling of Receptor Activator of Nuclear Factor-κB (RANK) Receptor Complex by Novel Osteoprotegerin-like Peptidomimetics Restores Bone Loss in Vivo" @default.
• W2073112480 cites W1513073936 @default.
• W2073112480 cites W1517494575 @default.
• W2073112480 cites W1650001464 @default.
• W2073112480 cites W1667210186 @default.
• W2073112480 cites W1677043489 @default.
• W2073112480 cites W1906075785 @default.
• W2073112480 cites W1937290251 @default.
• W2073112480 cites W1966041739 @default.
• W2073112480 cites W1971720206 @default.
• W2073112480 cites W1972001482 @default.
• W2073112480 cites W1978588499 @default.
• W2073112480 cites W1979639369 @default.
• W2073112480 cites W1986310612 @default.
• W2073112480 cites W1986784478 @default.
• W2073112480 cites W1987368791 @default.
• W2073112480 cites W1992159640 @default.
• W2073112480 cites W1992476947 @default.
• W2073112480 cites W1997323954 @default.
• W2073112480 cites W2000647744 @default.
• W2073112480 cites W2013645466 @default.
• W2073112480 cites W2015721537 @default.
• W2073112480 cites W2017086893 @default.
• W2073112480 cites W2019146941 @default.
• W2073112480 cites W2021154537 @default.
• W2073112480 cites W2026446264 @default.
• W2073112480 cites W2028421750 @default.
• W2073112480 cites W2028809209 @default.
• W2073112480 cites W2035104435 @default.
• W2073112480 cites W2035636765 @default.
• W2073112480 cites W2038204617 @default.
• W2073112480 cites W2039566310 @default.
• W2073112480 cites W2042274679 @default.
• W2073112480 cites W2042313745 @default.
• W2073112480 cites W2046382304 @default.
• W2073112480 cites W2049663776 @default.
• W2073112480 cites W2050408947 @default.
• W2073112480 cites W2056445524 @default.
• W2073112480 cites W2070869746 @default.
• W2073112480 cites W2072380594 @default.
• W2073112480 cites W2074661703 @default.
• W2073112480 cites W2076904776 @default.
• W2073112480 cites W2077710118 @default.
• W2073112480 cites W2081276820 @default.
• W2073112480 cites W2086937859 @default.
• W2073112480 cites W2097304380 @default.
• W2073112480 cites W2098085126 @default.
• W2073112480 cites W2103530688 @default.
• W2073112480 cites W2106433656 @default.
• W2073112480 cites W2106766464 @default.
• W2073112480 cites W2107201026 @default.
• W2073112480 cites W2108743612 @default.
• W2073112480 cites W2109079971 @default.
• W2073112480 cites W2113672914 @default.
• W2073112480 cites W2114935509 @default.
• W2073112480 cites W2116815941 @default.
• W2073112480 cites W2117710877 @default.
• W2073112480 cites W2117963070 @default.
• W2073112480 cites W2121778907 @default.
• W2073112480 cites W2131357215 @default.
• W2073112480 cites W2133081899 @default.
• W2073112480 cites W2134913715 @default.
• W2073112480 cites W2156467557 @default.
• W2073112480 cites W2167408727 @default.
• W2073112480 cites W2168451480 @default.
• W2073112480 cites W2169802771 @default.
• W2073112480 cites W2170384111 @default.
• W2073112480 cites W2470223656 @default.
• W2073112480 cites W3120153401 @default.
• W2073112480 cites W4254761345 @default.
• W2073112480 doi "https://doi.org/10.1074/jbc.m309690200" @default.
• W2073112480 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14679212" @default.
• W2073112480 hasPublicationYear "2004" @default.
• W2073112480 type Work @default.
• W2073112480 sameAs 2073112480 @default.
• W2073112480 citedByCount "84" @default.
• W2073112480 countsByYear W20731124802012 @default.
• W2073112480 countsByYear W20731124802013 @default.
• W2073112480 countsByYear W20731124802014 @default.
• W2073112480 countsByYear W20731124802015 @default.
• W2073112480 countsByYear W20731124802016 @default.
• W2073112480 countsByYear W20731124802017 @default.
• W2073112480 countsByYear W20731124802018 @default.
• W2073112480 countsByYear W20731124802019 @default.
• W2073112480 countsByYear W20731124802020 @default.
• W2073112480 countsByYear W20731124802021 @default.
• W2073112480 countsByYear W20731124802022 @default.
• W2073112480 countsByYear W20731124802023 @default.

• next