KR101180431B1 - Peptide Inhibiting RANKL-RANK Interaction and Pharmaceutical Composition Comprising the Same - Google Patents

Abstract

The present invention relates to novel peptides and their use which interfere with the interaction of ranks with rank ligands. By using the novel peptide and pharmaceutical composition comprising the same, which inhibits the interaction between the rank and the rank according to the present invention, it is possible to prevent or treat bone diseases caused by the differentiation and activation of the osteoclasts by inhibiting the differentiation of the osteoclasts. It works.

Description

Peptide Inhibiting RANKL-RANK Interaction and Pharmaceutical Composition Comprising the Same}

The present invention relates to novel peptides that inhibit the interaction of rank (RANKL) with rank (RANK) and uses thereof.

Bone is an important part of the body that supports the soft tissues and weight of the body, surrounds the internal organs, protects the internal organs from external shocks, structurally supports the muscles or organs, and stores calcium and other essential minerals in the body. It is a special organ maintained by the balance between bone resorption of bone through osteoclasts and bone formation through osteoblasts. The rank RANK (receptor activator of NF-kB), rank RANKL (RANKL) and osteoprotegerin (OPG) systems have led to breakthroughs in bone biology. The interaction between osteoblasts, stromal cells rank, and osteoclast rank receptors results in maturation of osteoclasts and subsequent resorption of bone. ^1-4 OPG functions as a water soluble inducer receptor for rankle and competes with rank to bind to rank. In other words, when the rankl binds to the rank, the receptor on the surface of the osteoclast progenitor cells, the osteoclast progenitor cells mature into osteoclasts and bone resorption occurs.Once OPG binds to the rankl, the binding between the rankl and the rank is blocked to form osteoclasts. This is suppressed and no more reabsorption of bone occurs. Therefore, OPG is in vivo ( in in vivo or in vitro ( in In vitro , it has been known as an effective inhibitor of osteoclast maturation and activation. ^{5-6 In} addition, osteoblasts / stromal cells and osteoclast precursors, rank / rank / OPG can be expressed in a variety of skin cells and therefore their role is associated with many other biological functions. Rankine / rank binding has been known to regulate mammary gland formation and temperature in women during lymph node organogenesis and pregnancy. ^7-10

The ratio between rank and OPG regulates bone metabolism by bone formation or resorption of bone. Therefore, if this ratio is not properly controlled, it causes an imbalance between bone formation and bone resorption and causes bone diseases such as osteoporosis, rheumatoid arthritis and bone deformity. The mutations of rank, OPG and rankl found in humans were found in ^11-14 autosomal recessive osteopetrosis (ARO ^{15 -16} ), expansile skeletal hyperphosphatasia (ESH ¹⁷ ), familial expansile osteolysis (FEO ¹⁸ ), early-onset Paget's disease ¹⁹ , adolescent par jetbyeong is associated with a rare genetic skeletal abnormalities such as ^{(Juvenile Paget's disease (JPD 20} -23)). Because the role of rank / rank / OPG proteins in bone metabolism is important, binding between them is considered to be an important target for suppressing bone metabolism-related diseases. Many therapies have been developed, such as OPG-Fc, RANK-Fc, anti-RANKL antibody, and RANKL vaccine, and ranks of OPG or TNF receptors that have the effect of interfering with the rankle signaling process. Peptides imitating binding loops (OP3-4 ^24-25 , WP9QY ²⁶ ) have been developed.

Ranke-rank belongs to the Tumor Necrosis Factor (TNF) Ligand-Receptor family, and these ligands and receptors have a similar mode of binding: in which the receptors are formed by the edge-to-face packing of the monomer subunits It binds to the groove of the binding site of the monomers of the ligand of the sieve. ^27-29 However, the structural features of the bonding surface that determine the binding of a particular ligand-receptor, in particular the manner of binding of the rankle-rank, have not been determined despite the crystal structure of the rankle being revealed.

The inventors of the present invention have uncovered structural determinants that determine specific ligand-receptor recognition, and as a result, continue to study bone-related diseases and provide molecular basis for the development of new drugs. In addition to identifying the crystal structure of the Rankine-Rank conjugate in 2.5 micron-sized mice, identifying the specific structure involved in the structure and binding of the Rankel-Rank conjugate, and a novel structure that suppresses the Rank-Rank interaction from the loop-3 structure. The invention was completed by developing the peptide (L3-3S).

Lacey, D. L., et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 93: 165-76. 2. Yasuda, H., et al. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin / osteoclastogenesis- inhibitory factor and is identical to TRANCE / RANKL. Proc. Natl. Acad. Sci. U. S. A. 95: 3597-602. 3. Boyle, W. J., Simonet, W. S., and Lacey, D. L. 2003. Osteoclast differentiation and activation. Nature. 423: 337-42. 4. Teitelbaum, S.L., and Ross, F.P. 2003. Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4: 638-49. 5. Simonet, W. S., et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 89: 309-19. 6. Yasuda, H., et al. 1998. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG / OCIF inhibits osteoclastogenesis in vitro. Endocrinology. 139: 1329-337. 7.Theill, L.E., Boyle, W.J., and Penninger, J.M. RANK-L AND RANK: T Cells, Bone Loss, and Mammalian Evolution. Annu. Rev. Immunol. 20: 795-823 8. Kong, Y.-Y., et al. 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 397: 315-23. 9. Fata, J.E., et al. 2000 The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103: 41-50. 10. Anderson, D. M., et al. 2009.Central control of fever and female body temperature by RANKL / RANK. Nature 462 (7272): 505-509. Tanaka, S., Nakamura, K., Takahasi, N., and Suda, T. 2005. Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL-RANK signaling system. Immunol. Rev. 208 (1): 30-49. 12. Vega, D., Maalouf, N.M., and Sakhaee, K. 2007. CLINICAL Review #: the role of receptor activator of nuclear factor-kappaB (RANK) / RANK ligand / osteoprotegerin: clinical implications. J. Clin. Endocrinol. Metab. 92 (12): 4514-21. 13. Theoleyre, S., Wittrant, Y., Tat, S.K., Fortun, Y., Redini, F., Heymann, D. 2004.The molecular triad OPG / RANK / RANKL: involvement in the orchestration of pathophysiological bone remodeling. Cytokine Growth Factor Rev. 15: 457-475. 14. Wright, H.L., McCarthy, H.S., Middleton, J., and Marshall, M.J. 2009. RANK, RANKL and osteoprotegerin in bone biology and disease. Curr. Rev. Musculoskelet. Med. 2 (1): 56-64. 15. Sobacchi, C., et al. 2007. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat. Genet. 39 (8): 960-962. 16. Guerrini, M. M., et al. 2008. Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations. Am. J. Hum. Genet. 83 (1): 64-76. 17. Whyte, M.P., and Huhges, A.E. 2002.Expansile skeletal hyperphosphatasia is caused by a 15-base pair tandem duplication in TNFRSF11A encoding RANK and is allelic to familial expansile osteolysis. J. Bone Miner. Res. 17: 26-9. 18. Hughes, A.E., et al. Mutations in the TNFRSF11A, affecting the signal peptide of RANK, cause familial expansile osteolysis. Nat. Genet. 24: 45-8. 19. Nakatsuka, K., Nishizawa, Y., and Ralston, S.H. 2003. Phenotypic characterization of early onset Paget's disease of bone caused by a 27-bp duplication in the TNFRSF11A gene. J. Bone Miner. Res. 18: 1381-385 20. Whyte, M. P., et al. 2002. Osteoprotegerin deficiency and Juvenile Paget's Disease. N. Engl. J. Med. 347: 175-4. 21. Chong, B., et al. Mutations in the gene encoding osteoprotegerin cause idiopathic hyperphosphatasia. J. Bone Miner. Res. 7 (suppl 1): S139. 22. Cundy, T., et al. A mutation in the gene TNFRSF11B encoding osteoprotegerin causes an idiopathic hyperphosphatasia phenotype. Hum. Mol. Genet. 11 (18): 2119-2127. 23. Middleton-Hardie C., et al. 2006. OPG causes juvenile Paget's disease by impairing both protein secretion and binding to RANKL. J. Bone Miner. Res. 21 (3): 438-45. 24. Cheng, X., et al. 2004. Disabling of receptor activator of nuclear factor-kappaB (RANK) receptor complex by novel osteoprotegerin-like peptidomimetics restores bone loss in vivo. J. Biol. Chem. 279 (9): 8269-77. 25. Heath, D. J., et al. 2007. An osteoprotegerin-like peptidomimetic inhibits osteoclastic bone resorption and osteolytic bone disease in myeloma Cancer Res. 67 (1): 202-8. 26. Aoki, K., et al. 2006. A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss. J. Clin. Invest. 116: 1525-534. 27. Banner, D. W., et al. 1993. Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex: Implications for TNF receptor activation. Cell 73: 431-445. 28. Mongkolsapaya, J., et al. 1999.Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation Nat. Struct. Biol. 6 (11): 1048-53. 29. Hymowitz, S.G., et al. 1999. Triggering cell death: the crystal structure of Apo2L / TRAIL in a complex with death receptor 5. Mol. Cell 4 (4): 563-71 30. Ito, S., et al. 2002.Crystal Structure of the Extracellular Domain of Mouse RANK Ligand at 2.2-A Resolution. J. Biol. Chem. 277 (8): 6631-6636. 31.Otwinowski, Z., and Minor, W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307-26. 32. Vagin, A., and Teplyakov, A. 2000. An approach to multi-copy search in molecular replacement. Acta Cryst. D 56: 1622-1624. 33.Murshudov, G.N., Vagin, A.A., and Dodson, E.J. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Sect. D 53: 240-55. 34. Emsley, P., and Cowtan, K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D 60: 2126-132. 35. Terwilliger, T.C. 2004. Using prime-and-switch phasing to reduce model bias in molecular replacement. Acta Crystallogr. Sect. D. 60, 2144-2149. 36.Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26: 283-91. 37. DeLano, W.L. 2002.The PyMOL molecular graphics system on world wide web http://www.pymol.org.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel peptide that can interact with the rankle, thereby inducing osteoclast differentiation by binding to the rank on the surface of the osteoclast differentiation cell and thus inhibiting the process of inducing bone disease. To prevent or treat a disease.

Still another object of the present invention is a nucleic acid encoding the peptide, a vector comprising the same, a recombinant microorganism transformed with the vector and a method for producing a peptide for inhibiting the interaction of the rank, characterized in that the culture of the recombinant microorganism To provide.

In order to achieve the above object, the present invention provides a novel peptide that interacts with the rankle to interfere with the rank and the binding of the rankle and consequently inhibits osteoclast differentiation by the rankle.

The present invention also provides a pharmaceutical composition for preventing or treating a bone disease comprising the peptide.

The present invention also provides nucleic acids encoding polypeptides that inhibit the interaction of ranks with ranks.

The present invention also provides a recombinant vector comprising a nucleic acid encoding a polypeptide that inhibits rank-rank interactions.

The present invention also provides a recombinant microorganism transformed with a recombinant vector comprising a nucleic acid encoding a polypeptide that inhibits rank-rank interaction.

The present invention provides a novel peptide that inhibits the interaction between the rank and rank, by inhibiting the differentiation of osteoclasts induced by the rankle to prevent or treat bone-related diseases caused by the differentiation and activation of osteoclasts It has the effect of providing a pharmaceutical composition.

1 relates to the overall structure of the eRANK, eRANKL and eRANK-eRANKL conjugates (tertiary structure of A: eRANK-eRANKL conjugate, binding surface of B: eRANK-eRANKL conjugate, C: free eRANKL and RANKL to which eRANK is bound Superposition, loop 1 (red), loop 2 (green), loop 3 (blue)), D: structure of RANK.
Figure 2 relates to the specific recognition sites of ligand-receptors (A: loop 1 (red), loop 2 (green), loop 3 (blue) of the rankle, B: the side of the rank and rank binding, C: rank and Electrostatic marking on the rank mating surface).
FIG. 3 shows a ball-and-stick model of the functional residues of loop 3. FIG.
Figure 4 shows the results of isothermal titration carolimetry (ITC) analysis of the binding force between eRANKL and wild-type eRANK, variants D85A, K97A, E126A.
Figure 5 shows the results of the wild type eRANK, variant D85A, K97A, E126A on the osteoclast differentiation inhibitory effect.
Figure 6 shows the osteoclast differentiation inhibitory effect of L2-1, L2-2, L3-1, L3-2, L3-3 and L3-3S (A: L2-1, L2-2, L3-1, Osteoclast differentiation inhibitory effect of L3-2, L3-3, B: osteoclast differentiation inhibitory effect of L3-3, L3-3S and OP3-4)
7 shows a schematic of eRANK, eRANKL and eRANK-eRANKL conjugates (A: eRANK-eRANKL conjugate, B: eRANKL trimer, C: eRANK).
Figure 8 shows the interaction between residues present on the binding surface of the eRANK-eRANKL conjugate (A: loop 1, B: loop 2, C: loop 3).
9 shows the results of ITC analysis of the binding force between eRANKL and variant C125A.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a peptide consisting of 13 amino acid sequences, wherein the amino acid sequence of 5-9 (sequence 5 to 9) is SDCEC, and the amino acid sequence of 11-13 (from amino acid 11) The sequence up to amino acid 13) is YRR and relates to a peptide that inhibits the interaction between rank and rank, characterized in that amino acids 2 and 10 are linked to each other to form a ring.

The sequence of the peptide consisting of 13 amino acid sequences according to the present invention is as follows.

number 1 No.2 number 3 4 times 5 times 6th 7th 8th No. 9 10th No. 11 12 times 13th amino acid end I end end S D C E C I Y R R A: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp. Tyr, Val
Me: Cys, Asp, Glu, Lys, Arg

In the present invention, the peptide may be a peptide that inhibits the interaction between the rank and rank, characterized in that having the amino acid sequence of SEQ ID NO: 1.

In the present invention, the peptide may be a peptide that inhibits the interaction between the rank and rank, characterized in that having the amino acid sequence of SEQ ID NO: 2.

YCWNSDCECCYRR (SEQ ID NO: 1)

YCSNSDCECCYRR (SEQ ID NO: 2)

In the present invention, the cyclization may be a peptide that inhibits the interaction between the rank and rank, characterized in that formed by cysteine (Cys) is located at amino acid positions 2 and 10.

In the present invention, the cyclization is formed by positioning aspartic acid (Asp) or glutamine (Glu) at amino acid position 2 and lysine (Lys) or arginine (Arg) at amino acid position 10. It may be a peptide that inhibits the interaction of the rank and rank.

In the present invention, the cyclization is formed by placing lysine (Lys) or arginine (Arg) at amino acid position 2, and aspartic acid (Asp) or glutamine (Glu) at position 10 amino acid. It may be a peptide that inhibits the interaction of the rank and rank.

In the present invention, the peptide may be a peptide that inhibits the interaction between the rank and rank, characterized in that 1 to 4 amino acids are further connected to the N terminal of amino acid 1 or C terminal of amino acid 13 , The added amino acids are 20 amino acids (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) It may be selected from the group consisting of.

In the present invention, the peptide that inhibits the rank interaction with the rank and binds to the rank includes both the SDCEC domain and the YRR domain, and amino acids 2 and 10 of the amino acid sequence form a cyclization. Seven amino acids are positioned between amino acids 2 and 10 to form a stable structure in which cyclization occurs. In an embodiment of the present invention, L3-2 contains the SDCEC and YRR domains involved in binding to the rankle and amino acid positions 2 and 10, similarly to peptide L3-3 having an inhibitory effect on osteoclast differentiation according to the content. Disulfide bonds are formed by the position of Cys, but six amino acids are positioned between two Cys amino acids, thereby preventing the formation of a stable structure. Thus, they have no inhibitory effect on osteoclast differentiation.

In the present invention, amino acids 1, 3 and 4 of the 13 amino acid sequence including the SDCEC domain and the YRR domain are 20 amino acids (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val). Although the examples describe only L3-3S and L3-3 as oligopeptides having an inhibitory effect on osteoclast differentiation, SEQ ID NO: 1 confirming the interaction of important residues forming rank-rank bonds. Since amino acids 1, 3, and 4 of the amino acid sequence of do not greatly participate in forming a bond with the rankle, they may be amino acids selected from the group consisting of 20 amino acids. In addition, the activity of the novel oligopeptides that can interact with the rankle is maintained by the formation of a three-dimensional structure by proper folding and the presence of important residues involved in binding to the rankle, thus greatly affecting the binding to the rankle. It is understood that amino acids 1, 3 and 4, which are not involved, have the same activity as the oligopeptides of SEQ ID NO: 1 because they contain the SDCEC and YRR domains and can maintain proper folding, whatever amino acids are present. Can be.

In the present invention, the cyclization of amino acids 2 and 10 of the peptide consisting of 13 amino acid sequences may be formed by disulfide bonds. Disulfide bonds are formed by positioning cysteines (Cys) at amino acid positions 2 and 10.

In the present invention, the cyclization of amino acids 2 and 10 of the peptide consisting of 13 amino acid sequences may also be formed by amide bonds. Aspartic acid (Asp) or glutamic acid (Glu), which are acidic amino acids, is located at amino acid position 2, and basic amino acid lysine (Lys) or arginine (Arg) is located at amino acid position, thereby Amide bonds are formed and cyclized, maintaining a more stable structure in vivo. In addition, the amide bond may be formed by placing a basic amino acid Lys or Arg at amino acid position 2 and an acidic amino acid Asp or Glu at amino acid position 10.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating bone diseases caused by differentiation and activation of osteoclasts, comprising the peptide and a pharmaceutically acceptable carrier.

In the present invention, the bone disease includes, but is not limited to, osteoporosis, osteoplastic insufficiency, hypercalcemia, osteomalacia, rheumatoid arthritis, Paget's disease, bone loss caused by cancer and bone necrosis.

In the present invention, the pharmaceutically acceptable carrier may include, but is not limited to, diluents, excipients, disintegrants and binders.

In the present invention, the composition may further include a lubricant, a humectant, a sweetener, a flavoring agent, a preservative, and the like.

Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred. As used herein, the term “parenteral” includes pia, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical compositions of the present invention may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Useful diluents for oral administration in capsule form include lactose and dried corn starch. The active ingredient is combined with emulsifiers and suspending agents when the aqueous suspension is administered orally. If desired, sweetening and / or flavoring and / or coloring agents may be added.

The pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed. Can be changed. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

Administering a peptide capable of binding to the rank according to the present invention, the rank and the peptide according to the present invention instead of binding to the rank and the rank, it is possible to suppress the interaction between the rank and rank, thus, osteoclasts Differentiation is suppressed to prevent or treat bone diseases caused by differentiation and activation of osteoclasts.

The peptides according to the present invention can be prepared by general chemical synthesis, such as, but not limited to, solid-phase peptide synthesis, and transformed with a recombinant vector containing a nucleic acid encoding the peptide. The peptide may be cultured to express the peptide, and then purified by conventional methods.

Therefore, in another aspect, the present invention is characterized by culturing a nucleic acid encoding a peptide that inhibits rank-rank interaction, a recombinant vector comprising the nucleic acid, a recombinant microorganism transformed with the recombinant vector, and the recombinant microorganism. It relates to a method for producing a peptide that suppresses the interaction between the rank and rank.

As used herein, the term "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site allowing the foreign DNA fragment to be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

"Transformation" of the present invention means that DNA is introduced into a host cell and DNA is replicable as a chromosomal factor or by chromosomal integration completion, which introduces an external DNA into a cell to cause an artificial genetic change . Generally, transformation methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, lithium acetate-DMSO method, and the transformed recombinant microorganism is DNA And high expression efficiency of the introduced DNA are usually used. Bacteria, yeast, fungi, etc. can be used as all microorganisms including prokaryotic and eukaryotic cells.

In an embodiment of the present invention, to confirm the structure of the rank-rank conjugate, the rat rank RANTO (RANK ectodomain) and the rank of RANKL ectodomain (RANKL ectodomain) were used, and "eRANK and eRANKL", respectively, rank of the ectodomain, rank Means ectodomain of, and for convenience eRANK and eRANKL are also expressed as rank and rank.

In the present invention, "cyclization" refers to making a chain compound into a cyclic compound.

In the present invention, "disulfide bond" refers to a covalent bond formed by oxidation (dehydrogenation) of two sulfur elements.

In the present invention, "amide bond" refers to a bond in which water is released from an amino group and a carboxy group.

Ranke - Rank  Structural analysis of the conjugate

In the present invention, in order to produce a peptide that inhibits the interaction between rank and rank was determined the structure of the rank and rank conjugate.

The structure of the rank-rankle conjugate was determined by a molecular replacement method using the crystal structure of the rankle as a search probe. The final model of the conjugate consisted of rank residues 161-315 and rank residues 35-198. The conjugate, approximately 60 μs x 70 μs x 100 μs, is a three-rank molecule containing four cysteine-rich domains (CRDs) inserted into three gaps formed at the subunit interface of the trimer rank. It confirmed that it was comprised (FIG. 1A, FIG. 1B, FIG. 7A). Like other TNF family ligands, the Rankine monomer consists of one jellyroll β-sandwich and two antiparallel β-pleated sheets, and the Rankel trimer itself The self-assembly is derived mainly by the conserved hydrophobic interaction established by aromatic residues and by hydrogen bonding of certain internal subunits (Figs. 1D, 7A and 9B). ). When the rank binds to the rankle, it was confirmed that the structure of AA ', CD and EF loops were changed and eventually modified the N- and C-terminus of strands D and E (FIG. 1C).

Rank  Structural analysis

In the present invention, the specific structure of the rank involved in the rank-rank interaction.

The rank included four CRDs sites (Fig. 1C, Fig. 9B) and confirmed that there are structural features that are distinct from other common TNF family receptors. ^27-29 Six conserved Cys residues form disulfide pairs in each CRD of the common TNF family of receptors, whereas the third and fourth Cys residues are absent in the rank CRD2-CRD4 ( 1C). In addition, an unusual disulfide bond Cys125-Cys127 was present in CRD3 (FIG. 1D), and this specific disulfide bond was expected to be involved in rank-rank recognition specificity.

Ranke - Rank  Major joins of mating faces ( bonding Confirmation of)

In the present invention, the couplings present in the coupling face of the rank-rank were examined.

In the rank-rank conjugate, it was confirmed that two loops of CDR2 (loop 1, loop 2) and one loop of CDR3 (loop 3) interact with the rankle (FIG. 2). Binding of the rank to the rankle reduces the solvent accessible surface area by 7680 Å ² , which corresponds to 15.4% of the total area of contact with the solution of the receptor and ligand in the unbound state. Charged residues of the receptor and ligand were found to provide an electrostatic network that determines the recognition specificity of the receptor-ligand (FIG. 2C).

In loop 1, Asp85 of rank interacts with lysine and hydrogen bond with Lys281 and Arg283 of strand F of rank, and Arg190 and Gly191 are backbone carbonyl groups of Pro74 in AA 'loop of rank. It further stabilizes the rank-rank bond by forming hydrogen bonds with each of side chains of Glu76 and Glu76 (FIG. 8A, Table 2). Furthermore, rank Asp75 forms ionic bonds and water-mediated hydrogen bonds with the Arg190 of rank, and Leu88 (eRANK) -His252 (eRANKL) and Leu89 (eRANK) -Gln302 (eRANKL) Van der Waals binding in Esso further enhances the recognition specificity of the conjugate (FIG. 8A, Table 2).

The three residues of loop 2 (Asp94, Gly96, Lys97) are in wide contact with the rankle, and broad interactions are formed between ranks Asp94 and Lys97 and ranks Arg222, Asp299 and Asp301, and Asp94 is an Arg222 and charge- Forming enhanced hydrogen bonds, Lys97 interacts with two acidic residues, Asp299 and Asp301. At the same time, Asp94 and Lys97 were involved in the intra-subunit salt bridge, which is important for determining the overall shape of loop 2. In addition, there are additional hydrogen bonds between the backbone carbonyl oxygen of Gly96, Lys97 and the backbone, side chain nitrogen of His224 (FIG. 8B, Table 2).

Loop 3 provided the largest mating face. The backbone carbonyl atoms of Tyr119 and Ser123 form hydrogen bonds with Glu225 and Lys180 of Ranke, respectively (FIG. 8C, Table 2), and the backbone carbonyl oxygen of side chain and rank Asp124 is His179 and salt bridge. , Lys180 and hydrogen bonds were formed respectively. The backbone carbonyl groups of Cys125 and Cys128 formed hydrogen bonds with Gln236 / Tyr240 and His224 of Ranke, respectively. Extensive hydrogen bonding, salt bridges were observed between ranks Arg129, Arg130 and ranks Glu225, Glu268, Asn266 (FIG. 8C, Table 2).

The most notable feature of loop 3 is the presence of a unique disulfide bond at the end of the loop formed between Cys125 and Cys127. This specific disulfide bond is an important determinant of the shape of loop 3 and allows Glu126 to interact extensively with many residues in the rankle. Glu126 not only interacts with Lys180 in the rankle, (FIG. 3, 14C), on one side form Lys256, Asp301, Asp303, and on the other side Asn253, Gln291 and bidentate water to form water-mediated hydrogen bonds there was. In addition, it was confirmed that Glu126 has a van der Waals bond with Tyr240 (FIG. 3).

Looking at how loops 1, 2, and 3 combine with the rankle, we found that loop 2 was relatively small in size, but it was widely combined with the rankle through Asp94, Gly96, and Lys97, while loop 3 provided the widest mating surface. He was forming the most bonds with Ranke. Based on this structural information, the inventors have constructed several peptides that are expected to be able to bind to the rankle from the amino acid sequences of loops 2 and 3, and form peptides (L2-1, L2-2) to form a shape similar to the rankle binding loop. , L3-2, L3-3, L3-3S) were cyclized (Table 4).

Key residues involved in rank-rank interactions Rank Ranke Type of combine

Loop 1


Pro74 Arg190 hydrogen bond Asp75 Arg190 ionic, hydrogen bond Glu76 Gly191 hydrogen bond Asp85 Lys281, Arg283 ionic, hydrogen bond Leu88 His252 hydrophobic Leu89 Gln302 hydrophobic
Loop 2
Asp94 Arg222 ionic, hydrogen bond Gly96 His224 hydrogen bond Lys97 Asp301, Asp299 ionic, hydrogen bond




Loop 3



Tyr119 Glu225 hydrogen bond Ser123 Lys180 hydrogen bond Asp124 His179, Lys180 ionic, hydrogen bond Cys125 Tyr240, Gln236 hydrogen bond Glu126 Lys180 ionic, hydrogen bond Glu126 Tyr240 hydrophobic Cys128 His224 hydrogen bond Arg129 Tyr234 hydrophobic Arg129 Glu268 ionic, hydrogen bond Arg130 Glu225, Asn266 ionic, hydrogen bond

Positioning mutations ( Site directed mutagenesis )

To induce mutations in the rank genes, point mutations of the rank genes were introduced using the Quick Change mutagenesis kit (Stratagene) and confirmed by DNA sequencing. Rank mutations were expressed and purified in the same way as wild-type ranks.

Ranke - Rank  Measurement of bonding force ITC )

The binding force between rank and rank or rank and rank variants was measured using isothermal titration calorimetry (ITC). Thermal changes were observed with the VP-ITC Micro Calorimeter (MicroCal Inc., MA, USA). The thermal change caused by the addition of the substrate solution was determined by least squares regression analysis (MicroCal Inc., MA, USA) to obtain dissociation constant ( Kd ) , enthalpy (ΔH) and entropy (ΔS). ) To suitably adjust.

Differentiation analysis of osteoclasts Osteoclst Differentiation Assay )

To determine the activity of rank and wild-type ranks and rank variants to differentiate osteoclasts, osteoclast differentiation assays were performed. The osteoclast precursors of mice were extracted and purified from the tibia and femur of 5-8 week old males. Osteoclast precursors were incubated in 96 well plates (1 × 10 ⁵ / ml, 200 μl / well) in α-MEM containing 30 ng / ml M-CSF, 10% FBS. Various concentrations of rank and rank were added and the cells were incubated at 37 ° C. in moist air containing 5% CO ₂ . Differentiation of osteoclast precursors in vitro (in In vitro , it was measured by the level of tartrate-resistant acid phosphatase (TRAP) activity. TRAP staining assay was performed using the substrate Naphthol AS phosphate in 100 mM sodium acetate (pH 5.2). TRAP-positive multinucleated cells were counted as osteoclast-like multinucleated cells under a microscope and repeated three times each to obtain statistical data.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

In particular, in the following examples, only the structure of the rank-rank crystals of the mouse was targeted, but the sequence is almost the same in humans and mice, and in particular, the sequence of the loop moieties involved in binding is completely the same. Of course, it is obvious that the rank-rank crystal structure of human can be predicted from the rank crystal structure and the osteoclast differentiation inhibitory effect can be obtained by applying the peptide and pharmaceutical composition according to the present invention to human. In addition, in the following examples, only the amino acid sequence of the novel peptides L3-3 and L3-3S and the osteoclast differentiation inhibitory effect of the peptide were confirmed, but those skilled in the art would degenerate using the amino acid sequence of the peptide according to the present invention without excessive experimental burden. (Degenerate) After preparing the primers, PCR using the prepared primers to obtain a gene fragment and a vector comprising the same, and further can select a suitable host cell to obtain a transformed microorganism.

Ranke Ectodomain (eRANKL) and Rank Ectodomain (eRANK)  Of the gene that encodes Cloning , eRANKL and eRANK  Expression and Purification of Proteins

In order to obtain rank-rank conjugates, mouse lancets (eRANKL, residues 157-316) and ranks (eRANK, residues 32-201) were expressed and purified in the same manner. The genes encoding rank and rank ectodomains were expressed in pVFT3S vector having a cleavage site of the protease Tobacco Etch Virus (TEV) between N-terminal 6His-thioredoxin (Trx) and the inserted cDNA. 0690230). The prepared 6His-Trx-eRANKL and 6His-Trx-eRANK were inducted with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) at 15 ^o C for 24 hours, followed by Escherichia coli It was expressed in BL21 (DE3) and Origami cells (Novagen, WI, USA), respectively. Each fusion protein was purified by metal affinity purification using a Ni-NTA column (GE Healthcare, Uppsala, Sweden) and digested by TEV protease. The digested rank and rank were returned to the NTA column, and the passed portion was superdex-200 gel filtration chromatography (GE Healthcare, Uppsala, Sweden) in equilibrium with the buffer solution (25 mM Tris-HCl (pH 7.5), 50 mM NaCl). ) Was injected. To obtain the rank-rank conjugate, the purified rank and rank were mixed at a ratio of 1: 5 and incubated overnight at room temperature. The rank-rank conjugate was further purified by Superdex-200 gel filtration chromatography.

2-1: Crystallization and Data Collection crystalization and data collection )

For crystallization experiments, purified rank-rank crystals were concentrated to 10 mg / ml using Amicon ultrafiltration devices or Centricon (10 kDa MW curoff; Millipore, Mass., USA). The first crystallization screening is performed using the microbatch method using Crystal Screen I, II, SalRX (Hampton Research, CA, USA) and Wizard I, II (Emerald Biostructures, WA, USA) crystallization screen kits. ) At 22 ° C. Protein drops were prepared by mixing 1 μl of reservoir solution and 1 μl of Rankine-Rank conjugate solution. Diffraction-quality crystals were obtained within one week from a stock solution containing 100 mM trisodium citrate (pH 5.6) and 1.2 M ammonium sulfate.

Diffraction experiments were performed on beam line 4A of Pohang Light Source (Korea) and AR-NW12 of Photon Factory (Japan). The final X-ray diffraction data was collected from Photon Factory (Japan) at minus 173 ° C. with an ADSC Quantum 210 CCD detector of beam line AR-NW12. Binder determination was determined in unit cell parameters, a = b = 120.86 mm 3, c = 93.43 mm 3; space group with α = β = 90 ^o and γ = 120 ^o Belonged to P3 . As a result, it was confirmed that two rank-rank conjugates exist in one unit cell. Diffraction data of the DENZO and SCALEPACK HKL program suite The program was processed and quantified. ³¹ Data statistics for synchrotron data collection and processing are shown in Table 3.

2-2: structure determination and purification ( structure determanation and refinement )

The crystal structure of the rank-rank conjugate was determined by the replacement of molecules using the rank of monomer (PDB ID 1iqa) as a search model. ³⁰ raengkeul two subunits was found using the MOLREP program by the rotating function (rotation function) and translation search (search translation). ^{32 After} several rounds of purification with REFMAC ³³ , an electron density map of the rank molecule was obtained. In addition to solvent flattening with the RESOLVE ³⁵ program, additional modeling and purification with COOT ³⁴ resulted in R values of 22.6 and R _free , respectively. The final model result was 24.9% (for 10% data samples). The refined structure was used with COOT and PROCHECK ³⁶ It was evaluated and refinement statistics are shown in Table 3. Figures of all structures were prepared using the PYMOL ³⁷ program.

Data collection and refinement statistics Data collection statistics Space group P 3 Unit cell parameters (Å) a = b = 120.86, c = 93.43 No. of molecules in AU 4 (two eRANKs, two eRANKLs) Wavelength (Å) 1.0000 Resolution range (Å) 50-2.5 Completeness (%) 100 (99.7) Unique reflections 52,575 (6,176) R _sym (%) ^a 8.9 (43.0)    I / σ (I) 31.4 (3.0) Redundancy 10.46 (7.40) Refinement statistics Resolution (Å) 50-2.5 R-work (%) ^b 22.7 R-free (%) ^c 24.9 No . of nonhydrogen atoms Protein atoms 4,936 Water molecules 182 RMS deviations Bond length (Å) 0.0095 Bond angles (°) 1.65 B factor Average main chain (Å ² ) 56.3 Average side chain (Å ² ) 57.8 Residues in Ramachandran plot (%) Most favored 84.1 Additional allowed 15.4 Generously allowed 0.5 Disallowed 0

^a Rsym = ∑ | Ⅰi- <Ⅰ> | / ∑ⅠX100

^b Rwork = ∑ || Fobs |-| Fcalc || / ∑ | Fobs |

^c Rfree = ∑ || Fobs |-| Fcalc || / ∑ | Fobs | (10% of the diffraction points obtained in the experiment were randomly selected and used to calculate Rfree)

Rank - Ranke  Functions involved in interaction Residue  Mutant In rank  Adhesion and osteoclast differentiation ability

Three substituents were mutated to alanine at the end of the three loops (D85 in Loop1; K97 in Loop2; E126A in Loop3) to identify the role of functional substituents causing ligand-receptor interactions. As a result of ITC measurement, the rank of the wild type showed a strong binding force (K _d = 230 nM) to the rank, while no interaction was observed between the variant E126A rank and the rank (FIG. 4). In addition, when wild-type ranks were injected, the signaling caused by the rank-rank interaction was inhibited to completely inhibit osteoclast formation, whereas the variant E126A rank did not bind to the rankle and thus, the rankle It was confirmed that it does not inhibit osteoclast differentiation induced by (Fig. 5). In the case of variant K97A, it could not bind to the rankle and inhibited the differentiation of osteoclasts (FIG. 4). However, in the case of variant D85A, binding to the rankle was maintained (K _{d =} 24.2 μM) and the differentiation of osteoclasts was also inhibited (FIGS. 4 and 5). Therefore, loop 1 is not as important as loop 2 or loop 3 to join the rankle, and loop 2 is small but plays a big role in joining. In addition, in the case of the variant C125A also did not bind to the rankle (Fig. 9), it was confirmed that the disulfide bond of Cys125-Cys127 is very important.

Polypeptide L2 -One, L2 -2, L3 -One, L3 -2, L3 -3 and L3 Manufacture of -3S

Several peptides were constructed from the amino acid sequences of loop 2 and loop 3 to provide therapeutics for bone diseases due to differentiation and activation of osteoclasts by interacting with and disrupting rank-rank interactions (FIG. 8). , Table 4). Cys and Tyr were introduced to make the cyclized peptides resembling the overall rank binding site. However, L3-1 did not introduce Cys or Tyr to prevent cyclization, and Cys128 was replaced with Ser to prevent unwanted disulfide bonds with Cys125 or Cys127. OP3-4, an OPG-mimic protein known to inhibit the formation of osteoclasts, was used as a control. ³⁴

Of the five peptides (L2-1, L2-2, L3-1, L3-2, L3-3) prepared, the effect of inhibiting osteoclast differentiation according to the content of only peptide L3-3 (dose-dependent inhibition) Is shown (FIG. 5B). L2-1 and L2-2 showed insignificant effects on the inhibition of osteoclast differentiation depending on the content, which means that loop 2 is less involved than loop 3 in the rank and rank binding. In addition, the fact that L3-1, which includes almost all sequences of loop 3 but only terminal disulfide bonds, does not show an osteoclast differentiation effect, suggests that modification by terminal disulfide bonds plays an important function. It also means that the limiting effect imposed on L3-3 stabilizes the structure of L3-3.

In order to increase the osteoclast differentiation inhibitory effect of L3-3, an experiment was carried out to replace the amino acid with another, and when the third Trp of the amino acid sequence of SEQ ID NO: 1 was replaced with Ser (Table 4), the effect was significantly increased. It was confirmed. The osteoclast differentiation inhibitory effect was increased by 20-40 times compared to L3-3 or OP3-4 (FIG. 6B). That is, 2.5 μM of L3-3S shows an effect corresponding to 100 μM of OP3-4 or L3-3. This result is because the Trp is replaced with Ser, which is structurally more stabilized, and the hydrophobicity of the peptide is increased by replacing the hydrophobic residue Trp with Ser, which is a polar residue, thereby increasing the activity of the peptide. to be.

Amino Acid Sequences of L2-1, L2-2, L3-1, L3-2, L3-3, L3-3S Amino acid sequence Amino acid sequence Loop 2
93- C D A GK ALV-100
L2-1 YC DAGKAL CY (SEQ ID NO: 3) L2-2 YC DGGKAL CY (SEQ ID NO: 4)
Loop 3


118-G Y HWN SDCEC C RR N-131

L3-1 GYHWNSDCECSRRN (SEQ ID NO: 5) L3-2 YC NSDCEC CY RR (SEQ ID NO: 6) L3-3 YC WNSDCEC CY RR (SEQ ID NO: 1) L3-3S YC SNSDCEC CY RR (SEQ ID NO: 2) OPG 113-LEIEFCLKHR- 122 OP3-4 YC EIEF CY LIR

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (15)

A peptide consisting of 13 amino acid sequences, wherein amino acid sequence 5-9 is SDCEC, amino acid sequence 11-13 is YRR, and sequence 2 and 10 are linked to each other to form a cyclization sequence Peptides that inhibit the rank interaction with the rank represented by the number 2.
delete delete delete delete delete delete Prevention of a bone disease selected from the group consisting of osteoporosis, osteoplasia, hypercalcemia, osteomalacia, rheumatoid arthritis, Paget's disease, bone loss due to cancer and bone necrosis, comprising the peptide of claim 1 and a pharmaceutically acceptable carrier Or therapeutic pharmaceutical compositions. delete delete delete delete delete delete delete

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