JP2002508160A - Receptor-derived peptides as modulators of receptor activity - Google Patents

Abstract

  (57) [Summary] Oligopeptides having an amino acid sequence corresponding to the extracellular domain of the receptor and a sequence similar to a regulatory peptide from an MHC class I antigen may enhance or reduce the physiological response of the ligand binding to the corresponding receptor. Take over. The oligopeptides are used in the diagnosis and treatment of diseases involving inadequate or inappropriate receptor responses, and in the screening of drug candidates that affect receptor surface expression. If the sequence corresponding to the regulatory peptide is modified or deleted, the modified receptor molecule is also useful for drug screening

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The field of the invention is the field of cell surface receptors, including the regulation of receptor internalization and the regulation of receptor activation in the presence or absence of ligands Regulation of the activity of

BACKGROUND OF THE INVENTION [0002] The complex regulatory balance between hormones, receptors and responding cells is important for the proper functionality of multicellular organisms. Sensitive environmental and genetic factors disrupt this balance and sometimes cause disease. The advent of molecular biology has meant that medically important hormones can be made available in therapeutically useful amounts. Among them are human growth hormone, EPO, TPO, insulin-like growth factor, insulin, epidermal growth factor and many others.

A condition of great economic and medical significance is insulin resistance, which is an essential feature of various clinical disorders, such as diabetes mellitus, obesity and certain types of hypertension. Individuals with non-insulin-dependent diabetes exhibit insulin resistance in peripheral tissues. They have subnormal glucose utilization in skeletal muscle, where glucose transport across the skeletal muscle cell membrane is the rate-limiting step in glucose metabolism. The defect may be in insulin-dependent glucose transport in diabetic skeletal muscle, where the glucose transporter 4 protein (GLUT
The level reduction of 4) is observed. In fat and muscle cells, insulin is
It stimulates a rapid and dramatic increase in glucose uptake, primarily by promoting redistribution of the GLUT4 glucose transporter from its intracellular storage site to the plasma membrane.

[0004] Insulin resistance can also be attributed to a deficiency in insulin action at the cellular level.
The insulin receptor is activated by binding of insulin to the α subunit of the receptor, which causes autophosphorylation of the intracellular β subunit region. Activated insulin receptors bind to cytosolic receptor substances that can influence the signaling cascade, resulting in a pleiotropic hormonal response. Most proteins involved in signal transduction pathways are not yet known, but each of them may play a role in various forms of insulin resistance. Heterogeneity in insulin resistance makes treatments that can act "upstream" of the signaling pathway very attractive, as many different medical conditions can be treated with a single drug.

[0005] Certain peptides have previously been shown to enhance the cellular response to certain hormones. This effect has been ascribed to the inhibition of the internalization of the corresponding hormone receptor. Insulin-stimulated glucose uptake is enhanced by adding peptides to responder cells, which offers the potential for improved treatment for insulin dependent and insulin resistant diabetes. Enhanced response can also be exploited for treatments involving other hormones. Improvements in the specificity of agents that enhance the activity of insulin and other hormones are of considerable interest because of their therapeutic benefits. The site of action of such a peptide on the receptor molecule is of interest for drug evaluation and design.

[0006] In addition, there is a great deal of interest in finding ligand substitutes or mimetics that can be used in place of natural ligands. This is of particular interest for ligand hormones, which may have multiple biological functions where only a small part is regulated. Related Literature Several groups have investigated the insulin receptor for residues involved in glucose transport and internalization. Rajagopalan et al. (1995, Bioc
Res. Commun. 211: 714-8) found that residue GPYL950-953 serves as a major endocytosis signal and that the sequence NPEY957-960 serves as a secondary signal. Levy-Toledano et al. (1993, Biochem. Biophys. Acta. 1220: 1-14
) Suggest that a structural domain located 43-113 amino acids from the C-terminus is required in intact cells for insulin-stimulated autophosphorylation and signal transduction. (1995, J. Cell Biol. 130: 1071-9) identified sequences involved in the differential acellular localization of glucose transporter isoforms and hormone responsiveness. The COOH-terminal 30 amino acids of GLUT4 are sufficient for its precise localization to an intracellular storage pool that migrates to the cell surface in response to insulin. further,
There is a report on important leucine residues in insulin receptor endocytosis (see Hamer et al., J. Biol. Chem. 272: 21685 (1997)).

[0007] US Pat. No. 5,385,888, issued Jan. 31, 1995, describes I class MHC peptide modulation of surface receptor activity. The data presented in International Patent Application PCT / US94 / O9189 suggest that these peptides must be biologically active in a regular conformation. The composition and use of such peptides is further described in International Patent Application PCT / US93 / 01758. Peptides are further described in International Application PCT / US89 / O
0876.

[0008] Regulation of receptor internalization by major histocompatibility complex I class molecules is derived from Olsson et al. (1994, Proc. Natl. Acad. Sci. 91: 9086-90) and from the insulin receptor. Regulation by peptides is described by Naranda et al., Proc. Natl.
Acad. Sci. 94: 11692 (1997). Peptides from the α1 domain of the major histocompatibility complex I class protein (MHC-1) suppress internalization of some receptors, thereby increasing the steady state number of active receptors on the cell surface. It has been suggested that MHC-1 is involved in regulating cell surface receptor activity. Stagsted et al. (1993, J. Biol. Chem.
268: 22809-13) demonstrate that such peptides inhibit the internalization of glucose transporter (GLUT4) and insulin-like growth factor II (IGF-II) in insulin-stimulated cells. .

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for modulating cell surface receptor activity. Accordingly, in one aspect, the invention provides a method of modulating the activity of a type 2 cell surface receptor containing an activating sequence, comprising binding an exogenous compound to the activating sequence. In one aspect, the binding is performed in the absence of any exogenous ligand that normally activates a cell surface receptor. In yet another aspect, the binding is
It is usually performed in the presence of a ligand that activates the receptor, in which case the level of activation is the same amount of ligand alone when using a combination of ligand and exogenous compound. Higher than you would.

[0010] In yet another aspect, the invention includes at least about 8 amino acids and less than about 40 amino acids, wherein the exogenous compound has an amino acid sequence corresponding to the activation sequence of the extracellular domain of a cell surface receptor. The present invention provides a method which is an oligopeptide. In another aspect, the present invention relates to a type 2 comprising an activation sequence on the surface of a mammalian cell.
Provided is a method of modulating the activity of a cell surface receptor, comprising binding an exogenous compound to an activating sequence.

In yet another aspect, the present invention is a method of treating a patient having a disease associated with a ligand that normally activates a type 2 cell surface receptor, comprising activating sequences of said receptor. Methods are provided that include administering an exogenous compound that binds. this is,
It can be done in the absence of an exogenous ligand, or in the presence of an exogenous ligand, wherein the level of activation, when using a combination of ligand and exogenous compound, Higher than with the same amount of ligand alone.

[0012] In yet another aspect, the invention adds a candidate bioactive agent to a target containing an activation sequence of a cell surface receptor, and said candidate bioactive agent is bound to the activation sequence. And a screening method including a step of determining whether or not the screening is performed. In a further aspect, the present invention is a screening method for a bioactive agent capable of modulating activation of a type 2 cell surface receptor, comprising a type 2 cell having an activating sequence.
Adding a candidate bioactive agent to a mammalian cell containing a cell surface receptor and determining whether the candidate bioactive agent binds to the activating sequence and modulates receptor activation. Methods for inclusion are provided.

Reference to database for nucleotide and amino acid sequences Complete mRNA encoding human insulin responsive glucose transporter (GLUT4)
The sequence has Genbank accession number M2O747 (Fukumoto et al. (1989) J. Biol.
Chem. 264: 7776-7779). The complete mRNA sequence encoding the human insulin receptor has Genbank accession number A18657 (International Patent Application WO / 91 // 17253). The complete mRNA encoding the human leptin receptor has Genbank accession number U43168 (Ta
rtaglia et al. (1995) Cell 83: 1263-1271). Human granulocyte colony stimulating factor (
G-CSF) has the EMBL accession numbers M5982O, M38O27, X5572O and X55721 (Larsen et al. (1990) J. Exp. Med. 172: 1559-1570). The complete sequence of the human interleukin 2 (IL-2) receptor is described in Swissprot Accession No. P01589.
(Leonard et al. (1984) Nature 311: 626-631). Human epidermal growth factor (
The complete sequence of the EGF) receptor has the Swissprot accession number P00533 (Ullrich et al.
l. (1984) Nature 309: 418-425). Additional Swissprot accession numbers are shown below (the remaining numbers are also readily available): interleukin-6 receptor, P08887; interleukin-8 receptor-B, P25025; interleukin-8 receptor-A, P240
24; Interleukin-11 receptor, U32324; Interleukin-12, P42701
Interleukin-17 receptor, U31993; EPO receptor, P19235; and TPO receptor, P40238.

[0014] Sequences for other cell surface receptors are known and can be readily ascertained by those skilled in the art. The sequences of known HLA and H-2 alleles can be found in Kabat et al. (1991) Sequences.
of Proteins of Immunological Interest, NIH publication no.91-3242, vo
l. 1, pp. 738-740, 761,770-771, 779-780 and 802-804.

DESCRIPTION OF SPECIFIC EMBODIMENTS Generally, the cell surface receptor in question is internalized or recycled into the cytoplasm in response to ligand binding. The present invention is based on the initial discovery that sequences of the extracellular portion of the cell surface receptor, called "internalization sequences" or "activation sequences," are involved in regulating the receptor response. Fortunately, the activating sequence is not directly involved in ligand binding. That is, the ligand binding site is remote from the receptor activation sequence.

Without being bound by theory, these activating sequences are important in two different directions: in regulating receptor internalization, and / or in regulating receptor activation. Initially, for some receptors, these activating sequences are likely to be involved in receptor internalization. That is, the addition of an oligopeptide corresponding to the activation sequence of the receptor may be, for example, to delay or inhibit internalization of the receptor (in some cases, as outlined below, the antagonist may be May occur), but may regulate receptor internalization. This suppression of receptor internalization may effectively provide more receptors at the cell surface. This increase or stabilization of the number of receptors on the cell surface can result in increased signaling per unit of ligand. This has therapeutic relevance for a number of disease states where reduced ligand binding or signaling is a problem, or where hormones are expensive or difficult to manufacture. For example, there are a number of diseases where hormonal sensitivity is reduced or hormone production is reduced, and where it is desirable to increase the efficiency of ligand signaling. Non-insulin dependent diabetes mellitus (NIDDM) is an example of such a condition.

Furthermore, for a particular type of cell surface receptor, referred to herein as “type 2 cell surface receptor”, the activating sequence upon activation of the receptor, ie, activation of the receptor's signaling pathway It turned out to be surprisingly important. Thus, oligopeptides corresponding to the activation sequence of the receptor may actually replace the requirement for a ligand and cause receptor activation even in the absence of a ligand . That is, even if the binding sites for the hormone ligand and the activating sequence are different, the addition of the activating sequence oligonucleotide will cause receptor activation. Without being bound by theory, type 2 cell surface receptors occur in monomeric units, each having a different ligand binding site and an internalization or activation site. In general, 2
The two monomeric receptors are held together by the binding of a single ligand molecule. This non-covalent "dimerization" activates the receptor and allows for downstream biological organs that are the result of ligand binding. Surprisingly, the present invention demonstrates that this dimerization and subsequent receptor activation can occur upon binding of the activating sequence oligopeptide in the absence of ligand . In addition, the action of the ligand and the action of the activating oligonucleotide are significantly synergistic, and may allow maximum signal with each reduced concentration. These oligonucleotides can therefore be utilized as ligand substitutes or to increase the response of a given amount of ligand. Further, as described in detail below, the present invention also allows for the generation of antagonists for receptor signaling.

Preferably, the internalization or activation peptide is derived from the sequence of the receptor to be modulated. The sequence corresponds to the region of the receptor on the extracellular surface, but usually does not directly participate in ligand binding, ie, no contact is made with the ligand (ie, the ligand's K Does not affect _D ). It should be noted that activating sequences from different receptors are highly specific, and generally that activating sequences from one receptor do not activate different receptors. The sequence of the receptor, as well as the positioning of the receptor in the cell membrane, is known in the art. Such information is accessible through public databases, as cited above.

In a preferred embodiment, the sequences and receptors described herein are from humans. However, as will be appreciated by those skilled in the art, the present invention may be useful in the creation and elucidation of animal models of human disease. Therefore, rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (cows, sheep, pigs, goats, etc.),
Can also be used. In addition, sequences from one organism can be tested in another organism. For example, rat sequences can be tested in humans and the like.

Accordingly, the present invention provides regulatory oligopeptides that include an activating sequence having an amino acid sequence that is at least substantially identical to the sequence of a portion of a cell surface receptor extracellular domain. In general, these oligopeptides also have sequences similar to the biologically active oligopeptides of the majority of histocompatibility locus class I antigens (US Pat. No. 5,385,888), the contents of which are incorporated herein by reference. include).
Oligopeptides modulate the action of ligands that bind to the corresponding receptor, thereby enhancing the physiological action of the ligand, and, as outlined above, by coordinating the receptor, It can act to completely replace child requirements.

The activation sequence is first identified by homology with the sequences of alpha ₁ domain of MHC class I antigens. MHC class I antigens include human MHC class I antigens and their mammalian equivalents, such as the class I antigens at the mouse H-2 locus, especially H-2D and K. Human MHC class I antigens include HLA-A, B and C.
Of particular interest are amino acids in the polymorphic region of the alpha-1 domain, especially amino acid 55
-90, usually 60-90, especially 62-90. region of the α-1 domain 60-85,
In particular, 62-85 or 72-82 have been found to be of particular interest. One MHC sequence of particular interest is ERETQIAKGNEQSFRVDLRTLLR (SEQ ID NO: 1, US Patent No. 5,385,888).
No.). Therefore, oligopeptides having sequences similar to these regions are preferred.

Using these sequences, in particular SEQ ID NO: 1, the sequence of the cell number receptor of any number is scanned for the same region. Suitable cell surface receptors include insulin receptor, insulin-like growth factor receptor, growth hormone receptor, glucose transporter (especially GLUT4 receptor), transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor Body, high density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptor such as IL-1, IL-2, IL-3, IL-4, IL-5
, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptor, human growth hormone receptor, VEGF receptor, PDGF Receptor, EPO receptor, TPO receptor, ciliary neurotrophic factor receptor, prolactin receptor and T cell receptor, but are not limited thereto. In addition, there are a number of "orphan" receptors whose biological functions have not yet been fully assigned, and are referred to herein as
Indicated by SwissProt reference number. Examples thereof include, but are not limited to: SwissProt ML1B (rat melanotin receptor 1)
SwissProt SCRC (human secretin receptor precursor); SwissProt NY1
R (Xenopus Xenopus neuropeptide Y receptor); SwissProt PAF
R (rat platelet activator receptor); and SwissProt BLR1 (Burkit lymphoma receptor for human, mouse and rat). Algorithms for sequence analysis are known in the art and include, but are not limited to: best fit sequence programs (Devereux with preferred default settings)
et al., Nucl. Acid Res. 12: 387-395 (1984)); BLAST (Altschul et al.,
(1990) J. Mol. Biol. 215: 403-10); ADVANCE and ADAM (Torelli and Robo).
tti (1994) Comput. Appl. Biosci. 10: 3-5); and FASTA (Pearson and
Lipman (1988) PNAS 85: 2444-8). Sequence similarity can be found in Wisconsin Package, ver
sion 8.0-Open VMS, can be determined using the Genetics Computer Group.

[0023] Preferably, the amino acid sequence of the receptor region has at least about 10% sequence identity, and often at least about 15-20% sequence identity. Sequence similarity is at least about 30%, preferably at least about 35%, and often is at least about 45-50%. The examples provide the results of an exemplary similarity search. Such exemplary activating amino acid sequences are set forth below, but are not limited to: TWLGRQGPEGPSSIPPGTLTTLW from human glucose transporter GLUT4 (SEQ ID NO: 2). It is 13% identical and 39% similar to SEQ ID NO: 1.

[0024] 2. KTDSQILKELEESSFRKTFEDYLH from the human insulin receptor (SEQ ID NO: 3).
It is 35% identical and 56% similar to SEQ ID NO: 1. 3. EAEAAVATQETSTVRLKVSSTAVRT from human LDL receptor (SEQ ID NO: 4). It is 16% identical and 88% similar to SEQ ID NO: 1. 4. KTEAEKQAEKEEAEYRKVFENFLH from human insulin-like growth factor receptor (SEQ ID NO: 5). It is 32% identical and 54% similar to SEQ ID NO: 1.

[0025] 5. KKENKIVPSKEIVWWMNLAEKIP from the human leptin receptor (SEQ ID NO: 6). It is 17% identical and 43% similar to SEQ ID NO: 1. 6. EKKPVPWESHNSSETCGLPTLVQTY from human GCSF receptor (SEQ ID NO: 7). 7. GPHCVKTCPAGVMGENNTLVWKY from human epidermal growth factor receptor (SEQ ID NO: 8)
. It is 17% identical and 43% similar to SEQ ID NO: 1.

[0026] 8. EYELQYKEVNETKWKMMDPILTTSVPVY from human growth factor receptor (SEQ ID NO: 9)
. It is 32% identical and 48% similar to SEQ ID NO: 1. 9. ARGGTLELRPRSRYRLQLRARLN from human thrombopoietin receptor (SEQ ID NO: 10). It is 22% identical and 43% similar to SEQ ID NO: 1. 10. QRVEILEGRTECVLSNLRGRTRY from human erythropoietin receptor (SEQ ID NO: 11). It is 26% identical and 43% similar to SEQ ID NO: 1.

11. EMQSPMQPVDQASLPGHCREPPPW from interleukin-2 (IL-2) receptor
(SEQ ID NO: 12). It is 12% identical and 37% similar to SEQ ID NO: 1. 12. DPDEGVAGAPTGSSPQPLQPL from IL-2 receptor β chain (SEQ ID NO: 13). It is 19% identical and 38% similar to SEQ ID NO: 1. 13. QEEGANTRAWRTSLLIALGTLL from Interleukin-3 (IL-3) (SEQ ID NO: 14). It is 27% identical and 50% similar to SEQ ID NO: 1.

[0028] 14. EPSLRIAASTLKSQISYRARVRAW from interleukin-4 (IL-4) receptor
AQCY (SEQ ID NO: 15). It is 28% identical and 52% similar to SEQ ID NO: 1. 15. DYETRITESKCVTILHKGFSASVRTILQ from Interleukin-5 (IL-5)
SEQ ID NO: 16). It is 30% identical and 52% similar to SEQ ID NO: 1. 16. PAQEVARGVLTSLPGDSVTL from interleukin-6 (IL-6) receptor (SEQ ID NO: 17). It is 30% identical and 50% similar to SEQ ID NO: 1.

[0029] 17. GKSNICVKVGEKSLTCKKIDLTTIVK from Interleukin-7 (IL-7) (SEQ ID NO: 18). It is 26% identical and 61% similar to SEQ ID NO: 1. 18. EDMGNNTANWRMLLRILPQSF from interleukin-8 (IL-8) receptor B
(SEQ ID NO: 19). It is 24% identical and 43% similar to SEQ ID NO: 1. 19. EVLGNDTAKWRMVLRILPHTF from interleukin-8 (IL-8) receptor A
(SEQ ID NO: 20). It is 19% identical and 52% similar to SEQ ID NO: 1.

20. ELDPGFIHEARLRVQMATL from interleukin-9 (IL-9) receptor (SEQ ID NO: 21). It is 32% identical and 63% similar to SEQ ID NO: 1. 21. EVITDAVAGLPHAVRVSARDFL from Interleukin-11 (SEQ ID NO: 22)
). It is 32% identical and 45% similar to SEQ ID NO: 1. 22. EQPTQLELPEGCQGLAPGTEVTYRLQLHML from Interleukin-12 (SEQ ID NO: 23). It is 43% identical and 67% similar to SEQ ID NO: 1.

23. EWSDKQCWEGEDLSKKTLLRFW from Interleukin-13 (SEQ ID NO: 24
). It is 27% identical and 59% similar to SEQ ID NO: 1. 24. KQDKKIAPETRRSIEVPLNERI from Interleukin-13 (second sequence)
SEQ ID NO: 25). It is 23% identical and 41% similar to SEQ ID NO: 1. 25. DPNITVETLEAHQLRVSFTLWNESTHYQILLTSF from Interleukin-17 (
SEQ ID NO: 26).

26. EITTDVEKIQEIRYRSKLKLI from human platelet-derived growth factor (PDGF) receptor
(SEQ ID NO: 27). 27. EARCDFCSNNEESFILDADSNM from human vascular endothelial growth factor (VEGF) receptor
SEQ ID NO: 28). It is 27% identical and 50% similar to SEQ ID NO: 1. 28. TWQTPSTWPDPESFPLKFFLRY from human ciliary neurotrophic factor receptor-α (SEQ ID NO: 29). It is 27% identical and 59% similar to SEQ ID NO: 1.

29. DSQTNVSQSKDSDVYITDKTVL from T cell receptor α chain (SEQ ID NO: 30). It is 14% identical and 50% similar to SEQ ID NO: 1. 30. EWTQDRAKPVTQIVSAEAWGRADC from T cell receptor β chain (SEQ ID NO: 31).
It is 17% identical and 37% similar to SEQ ID NO: 1. 31. SQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRH from T cell receptor gamma chain (SEQ ID NO: 32). It is 29% identical and 50% similar to SEQ ID NO: 1.

32. VHTEKVNMMSLTVLGLRMLF from T cell receptor δ chain (SEQ ID NO: 33). It is 37% identical and 58% similar to SEQ ID NO: 1. 33. EKTDRFVMKKLNDRVMRVEYHFLSPY from human transferrin receptor (SEQ ID NO: 34). It is 29% identical and 54% similar to SEQ ID NO: 1. 34. EWEIHFAGQQTEFKILSLHPGQKYL from human prolactin receptor (SEQ ID NO: 35). It is 20% identical and 48% similar to SEQ ID NO: 1.

In addition, as outlined below, there are a number of “orphan” receptors to which specific functions have not yet been assigned, and those activating sequences that may be included in the present invention are listed below: 35. ARRKAKAERKLRLRPSDLRSFLTMF from rat melatonin receptor type 1B (SEQ ID NO: 38).

36. KLRTQETRGNEVSHYKRLARSTLLLIP from human secretin receptor (SEQ ID NO: 39). 37. GKYVCLEDFPED from Xenopus neuropeptide Y receptor type 1
KRFLSYTTLLFIL (SEQ ID NO: 40). 38. SFRVDSEFRYT from rat platelet activating factor receptor (SEQ ID NO: 41).

39. CLNPMLYTFAGVKFRSDLSRLLTKL from human Burkitt lymphoma receptor (SEQ ID NO: 42). 40. CLNPMLYTFAGVKRFSDLSRLLTKL from mouse Burkitt lymphoma receptor (
SEQ ID NO: 43). 41. CLNPMLYTFAGVKRFSDLSRLLTKL from rat Burkitt lymphoma receptor (
SEQ ID NO: 44).

The receptor sequence, ie, the activation sequence, has at least 8
Amino acids, usually at least about 12 amino acids, more usually at least about 18
It includes amino acids, and less than about 40 amino acids, and more usually less than 30 amino acids. In a preferred embodiment, the oligopeptide corresponding to the activation sequence of the extracellular domain of a cell surface receptor is made synthetically or by recombinant means. "Corresponding" means herein that the oligopeptide is identical to all or part of the activation sequence, or that the oligopeptide has substantial homology to the activation sequence, i.e., as described below. The oligopeptide has an amino acid substitution,
Means that they may have insertions or deletions.

As will be appreciated by those skilled in the art, the activation sequence can be modified as a modified oligopeptide or as a modified receptor, where the receptor is made using the modified activation sequence. . In a preferred embodiment, the activation sequence of the regulatory oligopeptide is altered. Preferably, any modifications do not substantially alter the biological activity, ie, they do not inhibit or prevent activation internalization or assembly of the activating sequence for the corresponding receptor. This is easily tested using the binding assays described herein. For example, amino acid substitutions, insertions and deletions can be made.

[0040] In one embodiment, amino acid substitutions are made. Generally, residues that are critical for biological activity are not changed or are conservatively changed. Critical residues are revealed using known mutagenesis techniques, followed by activity or binding assays. For example, when using the scanning mutagenesis technique, a single amino acid residue in the activation sequence is modified by substitution with an aliphatic amino acid, such as serine, alanine, glycine, valine, and the like.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative. In general, these changes should be made to a few (a fe
Made on w) amino acids. However, in certain circumstances, larger changes may be tolerated. If minor changes in the characteristics of the oligopeptide are desired, substitutions will generally be made according to the following chart: Chart 1 Original Residues Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu Functional or immunological Substantial changes in identity can be made by selecting substitutions that are less conservative than those shown in Chart 1, but these are generally not preferred. For example, the structure of the polypeptide backbone in the region of change, eg, α
Helix or β-sheet structure; alterations or hydrophobicity of the molecule at the target site; In general, substitutions predicted to produce the greatest change in polypeptide properties include (a) when a hydrophilic residue, such as seryl or threonyl, is (by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl. (B) cysteine or proline is (substituted) by any other residue; (c) a residue having a positively charged side chain, such as lysyl, arginyl or histidyl is a negatively charged residue; For example, when substituted with (by) glutamyl or aspartyl; or (d) when a residue having a bulky side chain, such as phenylalanine, is (substituted) with one without a side chain, such as glycine.

In a preferred embodiment, no more than about three substitutions or deletions are made, and the changes are
Less than about 20%, usually less than about 10%, of the number of amino acids in the active motif, but in some cases, more changes can be made. In some cases, it may be desirable to create an antagonist peptide that binds to the receptor but does not activate it, in which case more changes may be made. In a preferred embodiment, the invention provides an oligopeptide having at least about 60% homology to the activating sequence of a nuclear receptor described herein, preferably at least about 75%, particularly at least about 75%. About 80% is preferred, and in some cases the identity is as high as 90-95 or 98%.

However, this can be higher if only non-deterministic residues are changed. Similarly, if the biological function of the activating sequence is reduced, the amount of change may be greater. Preferred are conservative substitutions, as known in the art, eg, in large hydrophobic groups: in isoleucine, leucine, valine and phenylalanine; between serine and threonine; glycine and alanine; asparagine and glutamine; aspartic acid And glutamic acid; or lysine, arginine and histidine.

In addition to modifications within the activation sequence, the oligopeptide may contain additional sequences, as will be appreciated by those skilled in the art. For example, oligopeptides provide 1) a convenient linking site, eg, cysteine or lysine; 2) enhance stability; 3) bind to a particular receptor; 4) provide a site-specific effect; 5) to provide ease of purification (eg, epitopes or purified (His ₆ ) tags); 6) to change physical properties (eg, solubility, charge, etc.); and 7) to stabilize configuration. Can be extended. The oligopeptide can be linked to the non-wild-type flanking region by a linking group or as a fusion protein covalently linked by a cysteine (disulfide) or peptide bond. Oligopeptides can be conjugated via various divalent agents such as maleimidobenzoic acid, methyldithioacetic acid, mercaptobenzoic acid, S-pyridyldithiopropionate, and the like. The oligopeptide may be linked to a single amino acid at the N- or C-terminus of the chain of amino acids, or may be linked internally. For example, a peptide of the invention can be covalently linked to an immunogenic protein, such as keyhole limpet hemocyanin, ovalbumin, etc., to promote antibody production against an oligopeptide of the invention.

In a preferred embodiment, the oligopeptide is shorter than indicated herein, ie, the residues from the N- or C-terminus of the oligopeptide are biologically active, preferably complete biological Can be deleted with the retention of objective activity. In some cases, internal residues can be removed from the oligopeptide. Generally, this is done by subsequently removing residues and assaying for the ability of the receptor to bind the activating sequence. Once binding has been established, activation can be assessed.

Alternatively, the oligopeptides of the invention can be expressed with other peptides or proteins, such as being part of an internal or N- or C-terminal chain. Various post-expression modifications are achieved. For example, by using an appropriate coding sequence, a peptide of the invention may be attached at one end to a lipid group and provide for farnesylation or prenylation such that it may be inserted into a lipid membrane, eg, into a liposome.

The oligopeptides of the invention can be modified by the addition of a chemical moiety or group.
For example, oligopeptides can be PEGylated, where polyethyleneoxy groups provide enhanced lifespan in the bloodstream. The oligopeptides of the present invention may further be combined with other proteins, such as Fc of the IgG isotype, to enhance complement fixation, or may be combined with toxins, such as ricin, albin, diphtheria toxin and the like, especially the A chain. Oligopeptides can be conjugated to antibodies for site-specific action. Regarding the joining technique, for example, U.S. Patent Nos. 3,817,837, 3,853,914, 3,853
0,752, 3,905,654, 4,156,081, 4,169,105 and 4,043,98
See No. 9 (the contents of which are incorporated herein by reference). The oligopeptide may be further labeled, as abbreviated herein.

[0048] Oligomers of the regulatory oligopeptides of the invention can also be made. For example, the oligopeptide for drug screening includes: 1) an oligopeptide having at least substantially the sequence of the receptor region; 2) the sequence of the receptor region and an organism of the major histocompatibility locus class I antigen. MHC / receptor oligopeptides having the amino acid sequence of the active oligopeptide; and 3) receptor-derived oligopeptide homodimers (generally as head-to-tail dimers. In this case, WO 96/15426 (especially
This description is incorporated herein by reference) and a spacer of one to three small neutral amino acids may be present between the two active peptide sequences, as generally described herein). But not limited thereto.

[0049] Once identified, oligopeptides that include an activation sequence can be prepared according to conventional techniques, for example, by synthesis (eg, using a Beckman 990 type peptide synthesizer or other commercially available synthesizer). Peptides were prepared by recombinant methods (Sambrook e
t al., Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring H
arbor, NY, 1989), or, for example, as a fusion protein with a protein that is one of the specific binding pairs, and subsequently engineered with an affinity reagent. Proteolytic cleavage at the site allows purification of the fusion protein to produce the desired peptide (eg,
Driscoll et al. (1993) J. Mol. Bio. 232: 342-350).

In a preferred embodiment, the activating sequence contained within the receptor can be altered to produce a modified receptor. In the modified form of the receptor, the sequence corresponding to the regulatory peptide (ie, the activation sequence) is inserted, substituted or deleted such that the ability of the receptor to internalize in response to ligand binding is altered. It contains. Modifications can include deletions or substitutions of the entire oligopeptide sequence or a portion thereof. Such substitutions also include scanning mutations as outlined above.

The modification is conveniently performed using recombinant DNA technology. The DNA sequence encoding the desired receptor can be obtained from various sources, or from a cDNA library derived from publicly available sequence information. In cloned gene
Techniques for in vitro mutagenesis are known. Methods for site-directed mutagenesis are described in Sambrook et al., Supra, pp. 15.3-15.108; Weiner et al. (1993
) Gene 126: 3541; Sayers et al. (1992) Biotechniques 13: 592-6; Jones and
Winistorfer (1992) Biotechniques 12: 528-30; Barton et al. (1990) Nuclei
c Acid Res. 18: 7349-55; Marotti and Tomich (1989) Gene Anal. Tech. 6: 67-
70 and Zhu (1989) Anal. Biochem. 177: 1204. For example, to delete a sequence, a primer is created that spans the region. Upon hybridization, the region to be deleted forms a single-stranded loop. The loop can be excised by nuclease digestion or extended from the primer using an appropriate polymerase.

For expression, the DNA sequence is inserted into a suitable expression vector, in which case the native transcription initiation region can be used, or the exogenous transcription initiation region,
That is, promoters other than those associated with normally occurring genes in the chromosome can be used. Promoters can be introduced by in vitro recombination methods or as a result of homologous integration of the sequence into the chromosome. A wide variety of transcription initiation regions are known for a wide variety of expression hosts. Generally, there will be a selectable marker that will work in the expression host. A promoter can be operably linked to the coding sequence of the gene to generate a translatable mRNA transcript. Expression vectors have convenient restriction sites located at the promoter sequence sensation to provide for insertion of a nucleic acid sequence encoding a heterologous protein. The promoter in a suitable expression vector can be constitutive or inducible. Expression vectors for the production of fusion proteins (here exogenous fusion proteins) provide additional functionality, ie, increased protein synthesis, stability, limited antiserum, reactivity with enzyme markers such as β-galactosidase, and the like. I do.

[0053] The expression vector is transformed into a host cell. Expression hosts include prokaryotes or eukaryotes, especially E. coli, Bacillus subtilis, yeast cells, mammalian cells such as COS and CHO cells, HeLa cells, L (tk), primary cultures, insect cells, Xenopus egg cells, and the like. I can do it. Particularly preferred host cells are mammalian cells. Oligopeptides and modified receptors, once created, find use in a number of applications.

In a preferred embodiment, the oligopeptide is used in a method for inhibiting the internalization of a cell surface receptor response in a mammalian cell. The method involves adding an oligopeptide as described herein to a mammalian cell that expresses a cell surface receptor. Upon addition (simultaneous or sequential) of the ligand that binds the receptor, the oligopeptide suppresses receptor internalization. Alternatively, for ligand-independent type 2 receptors, ligands need not be added to alter receptor internalization, as outlined below.

In a preferred embodiment, the oligopeptide is used in a method for activating a receptor. As mentioned above, for some receptors, the addition of the oligopeptide is
Supersedes the requirement for ligands to bind to affect receptor activation,
Or increase it. As outlined above, cell surface receptors are believed to fall into two general classes: type 1 and type 2 receptors. Type 1 receptors generally have two identical subunits, which are covalently or otherwise associated together in the absence of a binding ligand, which can However, it is essentially a preformed dimer. Type 1 receptors include the insulin receptor and the IGF receptor. However, type 2 receptors are generally in monomeric form, with one or more of them resulting in the binding of one ligand to each of the two monomers or, as shown in the present invention, multimerization. Receptor activation is achieved by the binding of the oligopeptide described above. Type 2 receptors include growth hormone receptors,
Leptin receptor, LDL receptor, GCSF receptor, interleukin receptor such as IL
-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-1
3, IL-15 and IL-17 receptors, EGF receptor, EPO receptor, TPO receptor, VE
GF receptors, PDGF receptors, T cell receptors, transferrin receptors, prolactin receptors and ciliary neurotrophic factor receptors. Therefore, the present invention
Provided are receptors having exogenous compounds attached to their activating sequence, particularly having binding oligopeptides as described herein.

As is known in the art, the monomeric nature of these type 2 receptors can result in exceptional saturation kinetics. For example, with respect to growth hormone, it is known that saturation leading to reduced activation can occur at high levels of administered growth hormone. This is probably because at high levels of ligand, each individual receptor monomer binds to a ligand molecule, probably due to the presence of only one ligand binding site per monomer receptor. And therefore are not put together in dimeric form, i.e., each monomer has a binding ligand, rather than two monomers sharing a ligand. It is.

Receptors can be classified as type 1 (ligand dependent) or type 2 (ligand independent) based on several tests. As noted above, type 2 receptors show reduced receptor activation at high levels of ligand. Type 2 receptors can also be classified as such using the present invention because the addition of the internalization oligopeptide of the present invention results in receptor activation even in the absence of ligand.

As discussed herein, oligopeptides based on the activation sequence of the type 2 cell surface receptor result in receptor activation even in the absence of ligand. It is important in this case to note that the binding site is different from the activating or activating sequence. Accordingly, the present invention provides a method for activating a type 2 cell surface receptor by binding an exogenous compound to an activating sequence of the receptor. As a result of this binding, the two monomeric receptors dimerize and activate the receptor.
However, in an alternative embodiment, activation may be blocked by binding an antagonistic exogenous agent to the activating sequence to block activation of the receptor. That is, certain agents block activation of the receptor, even in the presence of the ligand.

“Exogenous compound” as used herein means an endogenous compound produced endogenously from a cell or organism, ie, it is artificially introduced into a cell or organism. . Exogenous compounds include biologically active agents, such as chemical and small organic moieties, as described below; and the oligopeptides described herein,
It is not limited to these. Screening for other molecules that accomplish the same thing as the oligopeptides of the invention, as understood by those skilled in the art and described below, and the mechanism of binding localization and receptor activity through binding has been established. Is a routine. This may be by finding an exogenous agent that binds and activates at least two monomeric receptors simultaneously, or by binding an internalization (activating) sequence of the receptor to bind this binding agent. By finding agents that make multimers (ie, dimers) of Preferred embodiments utilize the oligopeptides of the invention as exogenous compounds.

“Receptor activation” or grammatical equivalents, as used herein, refers to a biological function associated with a ligand that binds a cell surface receptor. As will be appreciated by those skilled in the art, this will vary widely depending on the identity of the receptor. For example, cell surface receptors often undergo phosphorylation as a result of ligand binding, which is generally, but not always, the conformational change of the receptor as a result of ligand binding. by. Thus, "activation" encompasses a conformational change within the receptor or as a result of monomeric receptors becoming multimeric, which enables the receptor to promote signaling. I do. For example, this configuration change causes the receptor to phosphorylate or associate with a third molecule, which in turn causes phosphorylation of the receptor, a third molecule, or yet another molecule. In this way, signaling is achieved.

[0061] In a preferred embodiment, the exogenous compound is added to the cell in the absence of an exogenous ligand. “Exogenous ligand” as used herein means a ligand that is not endogenously produced by a cell or organism. That is, the exogenous ligand is introduced into a cell or organism. The composition of the ligand, whether endogenously generated or exogenously introduced, is generally the same, and the designation is independent of the composition, depending on the source of the ligand, Is understood. As will be appreciated by those skilled in the art, there may be endogenous ligands present. The exogenous compound acts as a ligand substitute, but it does not bind to the ligand binding site as a competitor, but rather binds to the receptor's activating sequence, as outlined herein. I do. The presence or absence of binding of the exogenous compound can be determined by various methods, as will be appreciated by those skilled in the art, for example, by labeling the exogenous compound and detecting the labeled receptor, known binding agents, For example, it can be tested by a competition assay using the oligopeptide of the present invention or a method using a modified receptor containing no activating sequence.

In a preferred embodiment, the exogenous compound is added to the receptor, usually in the presence of a ligand that activates the receptor. Furthermore, as before, there may be endogenous ligands present, or exogenous ligands added in addition to the exogenous compound. In this embodiment, the level of receptor activation is higher when using a combination of ligand and exogenous compound as compared to the same amount of ligand alone. Indeed, a synergistic effect is observed for some other receptors. That is, the action of the additional ligand and the exogenous compound (in this case, the oligopeptide of the present invention) is, as generally shown in the Examples and the drawings, less than that of the ligand alone or the exogenous compound alone. large. Without being bound by theory, this is due to the fact that the oligopeptide binds to and does not have a binding ligand, and possibly further delays internalization. Therefore, it is desirable to add some exogenous ligand with the exogenous compound of the present invention.

Thus, in a preferred embodiment, the oligopeptide is administered therapeutically.
The oligopeptides of the present invention act to enhance the cellular response to hormones that bind to surface membrane receptors corresponding to the oligopeptides, for example, the insulin receptor is enhanced by the oligopeptide of SEQ ID NO: 3 and glucose transport is 2 oligopeptides. Hormones such as insulin, insulin-like growth factor, human growth hormone, glucose transporter, transferrin, epidermal growth factor, EPO, TPO, low density lipoproteins, human growth hormone, and interleukins are referred to herein as "therapeutic." Called "hormonal hormone" or "ligand". Enhancement of the cellular response to therapeutic hormones by the oligopeptides of the present invention may be unresponsive to the effects of such hormones, eg, if they are resistant or, in the case of type 2 receptors, the oligopeptides are actually coordinated. It provides a means to improve patient response that may actually serve as a child replacement. This is particularly desirable in some situations where administration of a hormone ligand has undesirable side effects, such as in the case of growth hormone. The use of oligopeptides or other exogenous compounds may allow for receptor activation without significant side effects.

In one embodiment, the oligopeptides of the invention can be administered to a patient in need of an enhanced response to natural levels of a therapeutic hormone. Alternatively, the oligopeptide can be administered to a patient together with a therapeutic hormone.
Of particular interest is the treatment of insulin resistance, which may be related to a defect in the glucose transporter or in the cellular response to insulin. Administration of an oligopeptide of the invention improves response to insulin therapy. Similarly, enhancing the action of human growth hormone is of particular interest. Human growth hormone is commonly administered as an injectable drug in a number of situations. Alternative therapies can include increasing the endogenous hormone response.

Similarly, with respect to type 2 cell surface receptors, exogenous compounds, such as the oligopeptides of the present invention, may respond as described above as ligand substitutes or in a defined amount of ligand hormone response. To increase, it can be administered to a patient. Thus, the present invention provides a method of treating a patient having a disease associated with a ligand that normally activates the type 2 cell surface receptor. For example, all patients who have insufficient or no growth hormone or who respond poorly to normal levels of growth hormone will all bind to the activation sequence of the growth hormone receptor and have receptor activity Can be treated by the administration of exogenous compounds that cause the internalization and delayed receptor internalization. Similarly, other diseases such as obesity or leptin related weight problems can be treated. It should be noted that diseases associated with that therapy where there are not enough ligands present in the patient and there are too many ligands present in the patient can be treated with the exogenous compounds of the invention. is there. For example, if too many ligands are present in the patient, e.g., in the range where ligand saturation is observed, addition of an exogenous compound, e.g., an oligopeptide of the invention, will result in dimerization of the receptor. Make it possible
And activation can actually make the body a low ligand.

When the exogenous compound or oligopeptide is administered with an exogenous ligand,
As will be appreciated by those skilled in the art, administration can be simultaneous or sequential. For therapy, where an oligopeptide is used, the oligopeptide is administered locally or parenterally, e.g., by injection at a specific site, e.g., subcutaneously, intraperitoneally, intravenously, intranasally, transdermally, etc. obtain. Injectable formulations include physiologically acceptable media such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycol, and the like. Water-soluble preservatives used include sodium bisulfite,
Sodium thiosulfate, ascorbate, benzalkonium chloride,
Chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in individual amounts from about 0.001 to about 5% by weight, preferably from about 0.01 to about 2% by weight. Suitable aqueous buffers used are alkali or alkaline earth carbonates, phosphates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives, such as carboxymethylcellulose,
It can be used as a carrier in an amount of about 0.01 to about 5% by weight. The formulation will depend on the purpose of the formulation, the particular mode used to modulate receptor activity, the intended treatment, and the like. The formulation may include a patch, capsule, liposome, time delay coating, pill, or may be formulated in a pump for continuous administration. The specific dose is
It can be determined empirically according to known methods (eg, Harrison's, Principl
^{es of Internal Medicine, 11 th ed} . Braunwald et al. Ed, McGraw Hill Book
Co., New York, 1987).

In general, a therapeutically effective dose of an oligopeptide of the invention will range from about 0.005 to 10, more usually from about 0.01 to 1 mg / kg of host weight, preferably from about 0.1 to about 1 mg / kg,
For example, about 0.3 to about 0.9 is preferred for leptin receptor oligopeptides. Such a dose is usually sufficient, at least 50%, to enhance the action of the therapeutic hormone. Administration can often be several times daily, usually once or more daily, or sometimes weekly, depending on the level of drug being administered. Oligopeptides can be administered alone or in combination with therapeutic hormones. The hormone is usually administered at a therapeutically effective dose, or the dose is reduced by 50%, usually 25%, to compensate for oligopeptide enhancement. The host is any mammal,
For example, they can be domestic animals, pets, laboratory animals and primates, especially humans. The amount will generally be adjusted by the half-life of the peptide, in which case the lower part of the dose will be determined if the peptide exhibits half-life enhancement or a depot formulation, such as a matrix that retains the peptide over an extended period of time. For example, provided as a sustained release composition comprising particles introduced into a collagen matrix, the use of a pump in which the use of a pump continuously injects the peptide over an extended period of time above the substantially continuous rate of Heller (Biodeg.
radable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews i
n Therapeutic Drug Carrier Systems, Vol. 1, CRC Press, Boca Raton, FL, 19
87, pp. 39-90) describe encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13: 850-856 describes controlled drug release from hydrophobic polymers. .

In a preferred embodiment, oligopeptides, modified receptors and cells containing modified receptors are used in screening assays. The identification of the amino acid sequence in this region of the receptor allows the design of drug screening assays for compounds that modulate receptor internalization or serve as ligand substitutes for the type 2 receptor.

Drug screening assays utilize the sequence information and peptide compositions of the present invention, eg, proteins, oligopeptides and synthetic derivatives thereof, to identify agents that modulate cell surface receptor internalization, or In the case of type 2 receptors, it allows the identification of agents that serve as ligand substitutes. Drug candidates capable of modulating surface receptor internalization compete with the bioactive oligopeptide for association with the intact receptor or interfere with the binding of the oligopeptide to the receptor-derived oligopeptide of the invention Drug candidates are identified by first screening for potential.

Thus, in a preferred embodiment, the method comprises combining the activation sequence and a cell surface receptor containing the candidate bioactive agent, and determining the binding of the candidate agent to the activation sequence. By "cell surface receptor" is meant herein any number of cell surface receptors that are normally internalized upon ligand binding. Suitable cell surface receptors have been abbreviated above. Preferred embodiments utilize human cell surface receptors, but receptors for other mammals, eg, rodents (mouse, rat, hamster, guinea pig, etc.), and farm animals (cows, sheep, pigs, horses, etc.). Can also be used. This latter embodiment is chosen in the development of animal models of human disease. Included in the definition of cell surface receptor are amino acid substitutions, insertions or deletions of the native sequence. Preferably, they do not alter the biological activity of the receptor, but, as outlined herein, in some cases:
It is desirable to modify the biological activity of the receptor.

Further, a portion of a cell surface receptor is included within the definition of a cell surface receptor. That is, a full-length receptor, or a functional portion thereof, can be used. In a preferred embodiment, for at least the type 1 receptor, the functional domain includes at least a ligand binding domain and an activating sequence such that the conformational change that occurs upon ligand binding to the receptor still occurs. I do. However, as abbreviated herein, a functional domain for a type 2 receptor may include only active sequences.

In general, in a preferred embodiment of the methods herein, the cell surface receptor is non-diffusively bound to an insoluble support (eg, a microtiter plate, array, etc.) having an isolated specimen receiving area. . Insoluble supports are made from any composition to which the peptide or receptor can be bound, are easily separated from soluble material, and are otherwise compatible with all methods of screening.

The surface of such a support can be solid or porous and have any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays,
Membranes and beads. These are typically glass, plastic (
(Eg, polystyrene), polysaccharides, nylon or nitrocellulose, Teflon ™, and the like. Microtiter plates and arrays are particularly convenient because large numbers of assays can be performed simultaneously using small amounts of reagents and specimens. The particular mode of attachment of the peptide or other protein is not critical as long as it is compatible with the reagents and overall methods of the invention, retains the activity of the peptide, and is non-diffusible. Preferred methods of attachment include the use of antibodies (which do not sterically block the ligand binding site or activating sequence when the receptor is attached to the support), "sticky" or direct attachment to an ionic support. Bonding, chemical crosslinking, synthesis of receptors on the surface, and the like. After binding of the peptide or receptor, excess unbound material is removed by washing. The specimen receiving area is then blocked by incubation with calf serum albumin (BSA), casein or other harmless protein.

In a preferred embodiment, particularly with respect to type 1 receptor assays, ligands or analogs bound by cell surface receptors are also added to the assay. That is, when an insulin receptor is used, the ligand is insulin, and when a human growth hormone receptor is used, the ligand is human growth hormone. As will be appreciated by those skilled in the art, ligand analogs may also be used. In a preferred embodiment, this is not necessary, for example, when assaying for type 2 receptors.

[0075] Candidate bioactive agents are added to the assay. New binding agents include
Specific antibodies, non-natural binding agents identified on screens of chemical libraries,
Peptide analogs and the like. Of particular interest are screening assays for agents that have low toxicity to human cells. A wide variety of assays, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays,
Immunoassays for protein binding, functional assays (such as phosphorylation assays) and the like can be used for this.

The term “agent” or “exogeneous compound”, as used herein, directly or indirectly activates cell surface receptor internalization and / or activation. Describes any molecule, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., that has the ability to alter and may be present in response to or in the absence of ligand binding. In general, multiple assay mixtures are assayed in parallel with different agent concentrations to obtain differential responses to different concentrations. Typically, one of these concentrations, ie, zero concentration or below the level of detection, is used as a negative control.

[0077] Candidate agents include numerous chemical species, but typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 to less than about 2,500 daltons. Candidate agents include functional groups required for structural interactions with proteins, particularly hydrogen bonding, and typically include at least one amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two functional chemical groups. Including. Candidate agents often include carbocyclic or heterocyclic structures and / or aromatic or polyaromatic structures substituted by one or more of the above functional groups. Candidate agents are also found in biological molecules such as peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

[0078] Candidate agents are obtained from a wide variety of sources, such as libraries of synthetic or natural compounds. For example, a number of means are available for random and specific synthesis of a wide variety of organic compounds and biomolecules, such as expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. In addition, natural or synthetically produced libraries and compounds can be readily modified by conventional chemical, physical and biochemical means. Known pharmacological agents can be subjected to specific or random chemical modifications, such as acrylates, alkylations, esterifications, amidations to produce structural analogs.

Determining the binding of a candidate bioactive agent to a receptor can be done in a number of ways.
In a preferred embodiment, the candidate bioactive agent is labeled and directly bound. For example, this attaches all or part of the cell surface receptor to a solid support,
Add a candidate labeling agent (eg, fluorescent label), wash off excess reagents,
This can be done by determining if the label is present on a solid support. Various blocking and washing steps known in the art may be utilized.

“Labeled” as used herein refers to a label that provides a detectable signal, eg, a radioisotope, a fluorescent substance, an enzyme, an antibody, a particle such as a magnetic particle, a chemiluminescent substance, or a specific fluorescent substance. It means that the compound is directly or indirectly labeled with a binding molecule or the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and antidigoxin, and the like. For a specific binding member, the complementary member is usually labeled with a molecule that provides for detection, according to known techniques, as described above. The label provides, directly or indirectly, a detectable signal.

[0081] In some embodiments, only one of the components is labeled. For example, oligopeptides can be labeled with tyrosine using ¹²⁵ I (eg, human GLUT4, insulin, IGF-1, G-CSF, IL-2 and the activation sequence of the hGH receptor are all tyrosine residues Or labeled with a fluorophore. Alternatively, more than one component can be labeled with different labels, eg, with ¹²⁵ I for oligopeptides and with a fluorophore for candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agent is determined by using a competitive binding assay. In this embodiment, the competitor is an oligopeptide as described herein. In one embodiment, the candidate bioactive agent is labeled. The candidate bioactive agent or oligopeptide, if present, is first added to the receptor for a time sufficient to allow binding. Incubations can be performed at any temperature that promotes optimal activity, typically between 4 and 40 ° C. Incubation times are selected for optimal activity, but can be optimized to facilitate rapid, high-throughput screening. Typically, 0.1 to 1 hour is sufficient. Extra reagents are generally
Removed or washed off. Next, a second component is added and tracked to show binding in the presence or absence of the labeled component.

In a preferred embodiment, the oligopeptide is added first, followed by the candidate bioactive agent. Substitution of the oligopeptide is an indication that the candidate bioactive agent can bind to the activating sequence and thus modulate receptor internalization. Thus, for example, if the oligopeptide is labeled, the presence of the label in the wash solution indicates displacement by the agent. Alternatively, where the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is added first, incubated and washed, and then the oligopeptide is added. Absence of binding by the oligopeptide may indicate that the bioactive agent was bound to the receptor with higher affinity. Thus, when a candidate bioactive agent is labeled, the presence of the label on the support, when considered in conjunction with a lack of oligopeptide binding, allows the candidate agent to bind the activating sequence and internalize the receptor. It shows that the zation can be adjusted.

In a preferred embodiment, the method comprises the steps of: providing a cell surface receptor, a ligand bound to the receptor (
If necessary), and combining the oligopeptides as described herein,
Producing a test mixture. A candidate bioactive agent is added to the test mixture, and binding of the candidate bioactive agent to the receptor activation sequence is determined. In this embodiment, either the oligopeptide or the candidate bioactive agent or therapy is labeled, a preferred embodiment utilizes a labeled oligopeptide such that displacement of the label indicates binding by the candidate bioactive agent. .

[0086] In a preferred embodiment, the method includes a differential screen to identify bioactive agents that can modulate internalization or activate the receptor. In this embodiment, the method comprises the steps of: providing a cell surface receptor, a ligand (
If necessary), and combining oligopeptides. The secondary sample is
Includes candidate bioactive agents, cell surface receptors, ligands (if needed) and oligopeptides. The binding of the oligopeptide is determined for both specimens, and a change or difference in binding between the two specimens indicates the presence of an action node that binds the activating sequence and possibly may regulate receptor internalization. . That is, the agent may bind the activating sequence if the binding of the oligopeptide is different in the secondary sample relative to the primary sample.

Alternatively, a preferred embodiment is to differentially identify drug candidates that bind the native receptor but cannot bind the modified receptor, eg, those with deleted activation sequences. Use screening. The structure of the receptor sequence is modeled and used in rational drug design to synthesize agents that interact with the site. Drug candidates that affect receptor internalization or receptor activation include those agents with respect to their ability to enhance or reduce the effect of a receptor-derived oligopeptide of the invention on the internalization or activation of a selected surface receptor. It is also identified by screening.

Screening is performed to find agents that interfere with the association of the biologically active MHC-derived oligopeptide with the oligopeptide of the invention, wherein the agent modulates internalization or The oligopeptides of the invention can activate the resulting receptors. This is performed using the method described above, but replacing the oligopeptide with a sequence from the α1-domain of the MHC class I antigen, eg, SEQ ID NO: 1.

A drug candidate, as well as various concentrations of the receptor-derived oligopeptide of the invention, is added to each of the sample receiving regions containing the support-bound peptide. The added oligopeptide is substantially the same amino acid sequence as the oligopeptide bound to the indicator and is labeled. Positive controls for active peptide binding and active peptide competitive binding may include specimens containing labeled active peptide alone and mixtures of labeled and unlabeled active peptide, respectively. Specimens containing labeled active peptide and unlabeled inactive peptide that do not aggregate with the binding peptide serve as negative controls for competitive binding to the peptide. Preferably, all controls and test specimens are performed in at least triplicate to obtain statistically significant results. Incubation of the whole specimen is for a time sufficient for binding of the labeled active peptide to the support-bound peptide.
After incubation, all samples are washed free of non-specific binding substances and the amount of bound labeled peptide is determined. For example, if a radioactive label is used to label the peptide, the sample is counted in a scintillation counter to determine the amount of bound labeled peptide.

In test specimens containing drug candidates, the amount of labeled active peptide bound to the support-bound peptide or receptor is in the range of the value of the negative control specimen for competitive binding, and Significantly lower than the negative control sample, in which case the drug candidate in the test sample can successfully and competitively bind to the support-bound peptide. Such drug candidates capable of competitive binding can mediate the regulation of cell surface expression of the receptor.

[0091] A variety of other reagents can be included in the screening assays. Examples of these include salts that promote optimal protein-protein binding and / or reduce non-specific or background interactions, neutral proteins such as albumin,
And reagents such as detergents. Reagents that otherwise improve the efficacy of the assay, such as protease inhibitors, nuclease inhibitors and antimicrobial agents, can also be used.

The mixture of components can be added in any order that provides for the requisite binding. Screening for agents that activate type 2 receptors is performed in a similar manner as described above. Further, in some embodiments, using the same basic assay as described herein, but looking for agents that block activation and / or internalization, ie, antagonists, It is desirable to prevent receptor activation. For example, agents that bind to the active sequence but do not allow for internalization of the receptor are antagonists. However, as will be appreciated by those skilled in the art, the antagonists may be linked together, e.g., via a covalent bond,
The dimer here binds at least two receptors, thus providing a "dimer" of antagonists that can act here as an active agent. This can be done with a linker, as will be appreciated by those skilled in the art.

[0093] In a preferred embodiment, a method of screening is provided. In this embodiment, a candidate bioactive agent as described above is added to a type 2 cell surface receptor containing an activating sequence. Receptors can be on the surface of cells or in vitro
Be present in. The system is then assayed to determine whether the candidate bioactive agent binds the activating sequence and activates the receptor.

[0094] Similarly, a preferred embodiment provides a screening method comprising adding a candidate bioactive agent to a mammalian cell having a type 2 cell surface receptor containing an activating sequence. The system is then assayed to determine whether the candidate bioactive agent binds the activating sequence and activates the receptor. In this way, a bioactive agent is identified. Compounds having pharmacological activity may be capable of enhancing or preventing the internalization of cell surface receptors in response to ligand binding, or may serve as ligand replacements as described herein. obtain. Binding of the oligopeptide of the invention to the corresponding site on the receptor is such an activity that there is an ability to prevent binding of the oligopeptide of the invention to the cognate receptor. A compound having the desired pharmacological activity is administered to a host in a physiologically acceptable carrier, as described above. The agent can be administered in various ways, eg, orally, parenterally, eg, subcutaneously, intraperitoneally, intravenously.
Depending on the method of introduction, the compounds can be formulated in various ways. The concentration of the therapeutically active compound in the formulation can vary between about 0.1 and 100% by weight.

Pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, ointments, lotions and the like. Pharmaceutical grade organic or inorganic carriers and / or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically active compound. Diluents known in the art include aqueous media,
Vegetable and animal oils and fats. Stabilizers, wetting and emulsifying agents, salts to change the osmotic pressure, or buffers to maintain an appropriate pH value, and skin penetration enhancers can be used as adjuvants.

Thus, administration of such bioactive receptor-derived oligopeptides, oligopeptide homodimers and MHC / receptor oligopeptides to enhance the physiological effects of ligands binding to cell surface receptors Is provided. The methods find use in the diagnosis and treatment of diseases involving inappropriate or inappropriate receptor responses. The data show that receptor internalization is suppressed by the presence of the oligopeptides of the invention, thereby providing more receptors on the cell surface and increasing the effectiveness of ligand binding. Show.

In a preferred embodiment, the oligopeptides of the invention can be used to predict the function of an unknown receptor, the so-called “orphan receptor”. That is, for receptors for which no known function is associated, as will be appreciated by those skilled in the art,
It is possible to add an oligopeptide to the receptor in vitro and search for the effect. The following examples are provided to further illustrate the above-described method of the present invention and to describe the best mode intended to carry out various aspects of the present invention. These examples do not limit the scope of the invention in any way, but are provided for illustration. The references cited herein are incorporated herein by reference.

EXAMPLES Example 1 A sequence similarity comparison was performed to determine the regions on the ectodomain of cell surface receptors involved in receptor internalization. For comparison, the commercial Wisconsin
Package, version 8.0-Open VMS, Genetics Computer Group. The complete receptor sequence is described in “Database References for Nucleotide and Amino Aci
d Sequences ", as obtained above from public databases.

The similarity was measured by Dayhoff and was determined by Gribskov and Burgess (1986, Nucl. Ac.
ids Res. 14: 6745-6763), based on the evolutionary distance between amino acids. Smith and Waterman (1981, Advances in Applied Mathematics 2: 482-28
The "local homology" algorithm of 9) finds the best segment of similarity between two sequences.

A similarity search was performed between SEQ ID NO: 1 and the amino acid sequence of the cell surface receptor abbreviated herein to identify the regions as indicated in the table below.

[0101]

[Table 1]

[0102]

[Table 2]

Example 2 Methods Insulin receptor modification and expression. Database references, as well as Ebin
a et al. (1985) Human insulin receptor gene as described in Cell 40: 747-758 is transfected into HeLa cells by electroporation with a pCR3 expression vector (Invitrogen, Cat. No. K3000-01). did. The method of electroporation is Bogg
s et al. (1986) Ex. Hematol. 149: 988-994. In transfected cells, the receptor displays insulin-dependent internalization.

Using amplification primers spanning the region to be deleted, residues 713-740 (SEQ ID NO: 36:
(TKTDSQILKELEESSFRKLTFEDYLHNV) to create a mutant form of the insulin receptor by deletion. The deletion mutant mIR was transfected into HeLa cells and the internalization of the mIR was then examined. Measurement of IR internalization. Stagsted et al. (1990) Cell 62:29
Receptor internalization was performed substantially as described in 7-307. In short, as shown in Table 1, 50 μl of transfected cells
Incubation at 10 ⁶ cells / ml with 625 pM 1251-labeled insulin in the absence or presence of 10 μM peptide in a final volume of 100 μl in a shaking water bath at 37 ° C. The cells are then washed with 50 μl of KRHB (pH 7.2) (non-acidic wash).
Alternatively, the cells were diluted with 50 μl of KRHB (pH 2.0) (acid washing solution) and incubated on ice for 5 minutes. Cells were finally harvested by centrifugation on silicone oil and free and cell-associated radioactivity was measured.

Glucose transporter in fat cells. The biological activity of the peptide was determined as described above (Stagst
ed et al. (1991) J. Biol. Chem. 266: 12844-12847) as measured by their effect on glucose uptake in rat adipocytes. Briefly, rat adipocytes were harvested from the epididymal fat pad and 5% with a 10% (final) lipocrit.
Suspension in Krebs-Ringer HEPES buffer (KRH) containing 5% bovine serum albumin. Peptide action was measured in cells stimulated maximally with insulin (10 nM). After equilibration at 37 ° C. for 30 minutes, the plate was incubated at 37 ° C. for 30 minutes with buffer (basal), 10 nM insulin + peptide. ¹⁴ CD-glucose was added and the cells were incubated for an additional 30 minutes and harvested on oil. Dose - by measuring the biological activity by response curve, filled out EC ₅₀ values, the maximum enhancement of insulin action (the above maximum about 40% of the insulin alone) was considered as 100%. Most peptides are 30μ
Not tested at concentrations higher than M. Peptides that enhanced maximal insulin action by less than 20% at 30 μM were considered inactive.

Peptides. Applied Biosystems Model 430A peptide synthesizer t-Boc NMP / HO
On phenylacetamidomethyl (PAM) resin using the Bt protocol, or
Using the modified Fmoc / BOP protocol of the Milligen / Biosearch Model 9600 synthesizer
-The peptide was assembled stepwise on alkoxybenzyl alcohol (Wang). The desired peptide was confirmed by sequence analysis, amino acid composition and fast atom bombardment mass spectrometry. The peptide was activated by overnight incubation of a 1 mM stock solution in 0.1 M NaCl at 37 ° C. (Stagsted et al. (1991) J. Biol. Chem.
266: 12844-12847).

Results Effect of peptide on receptor internalization. The kinetics of internalization for the insulin receptor and the mutated insulin receptor were determined in the absence or presence of the peptides: SEQ ID NO: 3, KTDSQILKELEESSFRKTFEDYLH (pepIR) and SEQ ID NO: 37, GNEQSFRVDLRTLLRYAGGGNEQSFRVDLRTLLRYA (DS-A85) . The data is shown in Table 2, where the numbers indicate% internalized receptor.

[0108]

[Table 3]

The data show that the mutated insulin receptor mIR does not internalize upon insulin binding, while more than 50% of the wild-type IR internalizes within 30 minutes. The pepIR peptide suppresses receptor internalization to the same extent as DS-A85. Effect of peptides on glucose uptake. With maximum insulin stimulation, pepIR
Did not significantly affect glucose uptake, indicating that pepIR
Does not affect GLUT4 internalization. Glucose uptake is increased 14 ± 3 fold with the addition of 10 μM insulin. Insulin +
10 μM DS-A85 peptide increased glucose uptake by 22 ± 4 fold, whereas the addition of insulin + 10 μM pepIR increased glucose uptake by 12 ± 4 fold, and the results were not significantly different from insulin alone.

GLUT4pep (SEQ ID NO: 2), at a concentration of 10 μM, does not affect insulin receptor internalization by transfected cells. In the presence of peptide, the percentage of internalized or receptor is 6 ± 9, and in the absence of peptide, it is 64 ± 7. The peptide suppresses GLUT4 internalization, as shown by its effect on glucose uptake upon maximal insulin stimulation. In the presence of 10 nM insulin, the enhancement of glucose uptake was 12 ± 4 fold. The enhancement was increased by a factor of 24 ± 2 with the addition of GLUT4pep. Thus, the peptide appears to inhibit GLUT4 internalization, but not insulin receptor internalization.

Oligopeptides having the sequence of the extracellular domain of a cell surface receptor and having sequence identity to the MHC class I antigen region are effective in inhibiting the internalization of the corresponding receptor It is clear from the above results.
Peptides enhance the cellular response to hormones like insulin,
It is therapeutically useful.

Example 2 Ligand Substitution and Synergistic Addition Protocol for Protein Phosphorylation and Activation of the Cognate Receptor Signaling Cascade Materials Suitable cell lines have the following attributes: they respond to the appropriate hormones However, they express cognate receptors, which carry molecules necessary for downstream signaling of hormone-induced responses. The growth medium is as recommended for the cell line.

Antibodies: Immunoprecipitation: 2-5 μg of anti-PY-99 (Santa Cruz Biotechnology), anti-receptor or signaling molecule specific antibody (eg Jak, STAT); Western blot: anti-receptor specific antibody , Signaling protein specific antibody (Santa Cr
uz Biotechnology), or anti-Ptyr (4G10, Upstate Biotech); the secondary antibody is filled with the primary antibody. Immunoprecipitation method: Protein G beads (Pharmacia)
20-40 μl of slurry.

Protease inhibitor mix in water (100 ×); 1 mg / ml aprotinin, 0.1 mg / ml
ml of pepsatin A, 0.1 mg / ml leupeptin, 0.1 mg / ml chymostatin, 0.1 mg / ml
ml AEBSF. Cell wash buffer: PBS containing 1 mM ortho-vanadate, 50 mM NaF, 20 mM β-glycerophosphate and 2 mM sodium phosphate.

Lysis buffer: 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 ×
Protease inhibitor mix, 50 mM NaF, 5 mM EDTA and 2 mM sodium phosphate. Blocking buffer: Blotto: TST (0.01 M Tris, pH 7.4, 0.15 M NaCl, 0.07
5% Tween 20 ™, 0.02% NaN ₃ ) + 0.5% dry skim milk powder.

Procedure Treatment of Cells Cells were grown to a density of approximately 1 × 10 ⁶ cells, spun down, and resuspended in the recommended medium for growth. Cells are incubated in serum-free medium at 37 ° C. for 14-18 hours (
5% CO ₂ ) starved, centrifuged and resuspended cells in serum free medium at a density of 1 × 10 ⁶ cells / ml. 10 ml of cell suspension was used per P100 tissue culture dish. Cell suspension with 0.01-30 μM peptide at 37 ° C. for 15-30 hours (5% CO ₂ )
Treated; (dissolve peptide just prior to assay). 10 ml of ice-cold cell wash buffer was added to the dish, transferred to a Falcon tube and quickly centrifuged at 4 ° C and 3000 rpm. The medium was carefully aspirated and the washing was repeated at 4 ° C.

The following steps were all performed on ice: the medium was aspirated and 0.6 ml of 2x lysis buffer / tube was added. This was pipetted up and down, transferred to an Eppendorf tube, left on ice for about 30 minutes, and centrifuged at 14,000 × g at 4 ° C. for 10 minutes. The supernatant (lysate) was used for immunoprecipitation. Immunoprecipitation: Protein G is washed with 0.5 × lysis buffer in Eppendorf tubes, added with 1-4 μg of anti-PY-99 antibody, and receptor-specific or signaling molecule (JAK2, STAT5) specific antibody Was added to the IP. The tubes were incubated for 2 hours at room temperature while rotating up and down. Add the lysate and rotate the test tube up and down 4 ° C
Overnight. The beads are washed three times with 1 × lysis buffer and 40 μl of SD
S-Sample buffer was added and the samples were boiled for 3 minutes.

Western blot analysis: Approximately 12 μl / specimen was run on an 8% polyacrylamide minigel, transferred to PVDF membrane (Millipore), blocked for 1 hour in blocking buffer, and the appropriate primary antibody 1: 1000, Incubated at 4 ° C. with o / n. The membrane was washed and incubated for 2 hours at room temperature with the appropriate secondary antibody-alkaline phosphatase conjugated secondary antibody 1: 2000. The PVDF membrane was washed with Blotto and developed on NBT / BCIP.

Protocol for EPO-R Activation (Phosphorylation) Using TF-1 cells from ATCC, the growth medium was 1 × penicillin / streptomycin, 2 mM glutamine, 10% fetal calf serum (Hyclone) and 1 ng / h RPM1 1640 containing ml of GM-CSF. Treatment of cells: Cells were grown to a density of about 1 × 10 ⁶ cells, spun down by centrifugation and resuspended in medium containing 5% serum and no GM-CSF. Cells at 37 ° C
The cells were starved for 14-18 hours (5% CO ₂ ), centrifuged and the cells resuspended at a density of 1 × 10 ⁶ cells / ml in medium without serum and GM-CSF. 10 ml of cell suspension was used per P100 tissue culture dish. Cell suspension with 1-30 μM peptide at 37 ° C for 15
30 hours (5% CO ₂₎ was treated; (dissolving peptide immediately before the test). 10 ml of ice-cold cell wash buffer was added to the dish, transferred to a Falcon tube and quickly centrifuged at 4 ° C and 3000 rpm. The medium was carefully aspirated and the washing was repeated at 4 ° C. The following steps were performed on ice: the medium was aspirated and 0.6 ml of 2x lysis buffer / tube was added.
This was processed up and down with a pipette, transferred to an Eppendorf tube, and left on ice for about 30 minutes. Centrifuge at 14,000 xg at 4 ° C for 10 minutes, remove supernatant (lysate), and remove anti-EP
O-receptor antibodies (Santa Cruz Biotechnology) were used for immunoprecipitation and Western blot as described above.

The results are shown in the figure. All publications and patent applications cited herein are incorporated by reference, as if each individual publication and patent application was specifically and individually indicated to be incorporated by reference. Included herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example to enhance understanding, it will be understood that certain changes and modifications may be made without departing from the spirit and scope of the appended claims. It will be readily apparent to one skilled in the art in light of the teachings of the present invention.

[0121]

[Sequence list]

 (2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecular type : Protein (xi) SEQ ID NO: 1:

[0122]

[Table 4]

(2) Information about SEQ ID NO: 2 (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 2:

[0124]

[Table 5]

(2) Information on SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 24 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 3:

[0126]

[Table 6]

(2) Information on SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 4:

[0128]

[Table 7]

(2) Information on SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 24 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 5:

[0130]

[Table 8]

(2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 6:

[0132]

[Table 9]

(2) Information on SEQ ID NO: 7: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 7:

[0134]

[Table 10]

(2) Information on SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 8:

[0136]

[Table 11]

(2) Information on SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 9:

[0138]

[Table 12]

(2) Information about SEQ ID NO: 10 (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 10

[0140]

[Table 13]

(2) Information on SEQ ID NO: 11: (i) Sequence characteristics: (A) Length: 23 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 11

[0142]

[Table 14]

(2) Information on SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 24 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 12:

[0144]

[Table 15]

(2) Information on SEQ ID NO: 13: (i) Sequence characteristics: (A) Length: 21 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 13

[0146]

[Table 16]

(2) Information on SEQ ID NO: 14: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 14:

[0148]

[Table 17]

(2) Information on SEQ ID NO: 15: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 15

[0150]

[Table 18]

(2) Information on SEQ ID NO: 16: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 16

[0152]

[Table 19]

(2) Information about SEQ ID NO: 17: (i) Sequence characteristics: (A) Length: 20 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 17

[0154]

[Table 20]

(2) Information on SEQ ID NO: 18: (i) Sequence characteristics: (A) Length: 26 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 18:

[0156]

[Table 21]

(2) Information about SEQ ID NO: 19: (i) Sequence characteristics: (A) Length: 21 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 19:

[0158]

[Table 22]

(2) Information about SEQ ID NO: 20: (i) Sequence characteristics: (A) Length: 21 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 20:

[0160]

[Table 23]

(2) Information on SEQ ID NO: 21: (i) Sequence characteristics: (A) Length: 19 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 21:

[0162]

[Table 24]

(2) Information on SEQ ID NO: 22: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 22

[0164]

[Table 25]

(2) Information on SEQ ID NO: 23: (i) Sequence characteristics: (A) Length: 30 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 23

[0166]

[Table 26]

(2) Information about SEQ ID NO: 24: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 24:

[0168]

[Table 27]

(2) Information about SEQ ID NO: 25: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 25

[0170]

[Table 28]

(2) Information on SEQ ID NO: 26: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 26:

[0172]

[Table 29]

(2) Information on SEQ ID NO: 27: (i) Sequence characteristics: (A) Length: 21 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 27:

[0174]

[Table 30]

(2) Information about SEQ ID NO: 28: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 28

[0176]

[Table 31]

(2) Information about SEQ ID NO: 29: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 29

[0178]

[Table 32]

(2) Information on SEQ ID NO: 30: (i) Sequence characteristics: (A) Length: 22 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 30:

[0180]

[Table 33]

(2) Information on SEQ ID NO: 31: (i) Sequence characteristics: (A) Length: 24 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 31

[0182]

[Table 34]

(2) Information on SEQ ID NO: 32: (i) Sequence characteristics: (A) Length: 36 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 32

[0184]

[Table 35]

(2) Information about SEQ ID NO: 33: (i) Sequence characteristics: (A) Length: 20 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 33

[0186]

[Table 36]

(2) Information on SEQ ID NO: 34: (i) Sequence characteristics: (A) Length: 26 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 34:

[0188]

[Table 37]

(2) Information on SEQ ID NO: 35: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 35

[0190]

[Table 38]

(2) Information on SEQ ID NO: 36: (i) Sequence characteristics: (A) Length: 27 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 36:

[0192]

[Table 39]

(2) Information on SEQ ID NO: 37: (i) Sequence characteristics: (A) Length: 36 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 37

[0194]

[Table 40]

(2) Information on SEQ ID NO: 38: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 38:

[0196]

[Table 41]

(2) Information about SEQ ID NO: 39: (i) Sequence characteristics: (A) Length: 27 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 39:

[0198]

[Table 42]

(2) Information about SEQ ID NO: 40: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 40:

[0200]

[Table 43]

(2) Information on SEQ ID NO: 41: (i) Sequence characteristics: (A) Length: 11 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 41

[0202]

[Table 44]

(2) Information on SEQ ID NO: 42: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 42:

[0204]

[Table 45]

(2) Information on SEQ ID NO: 43: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 43

[0206]

[Table 46]

(2) Information on SEQ ID NO: 44: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) ) Molecular type: protein (xi) SEQ ID NO: 44

[0208]

[Table 47]

[Sequence list]

[Brief description of the drawings]

FIGS. 1A and 1B: As measured by phosphorylation of the EPO receptor (EPO-R).
A synergistic effect of an EPO receptor-derived peptide (EPO-Rp, SEQ ID NO: 11) on the action of EPO. IP, immunoprecipitation; WB, Western blot. 1A and 1B are digital images of a Western blot. Before dissolution and IP and WB,
Cells were incubated with EPO and / or EPO-Rp for 20 minutes. The panel shows that EPO-Rp in itself stimulates EPO-R, but at 0.01 U / ml
EPO alone has a certain effect and 10 nM EPO-Rp has a weak effect, so it also shows that EPO-Rp has a strong synergistic effect, but 0.01 U / ml EPO + 10 nM EPO-Rp has a strong effect (FIG. 1B right). Thus, the results indicate that EPO-Rp can potently enhance endogenous levels of EPO, for example, in patients with renal disease where EPO production is significantly reduced.

FIG. 2: Kinetics of the action of EPO and EPO receptor-derived peptide (EPO-Rp, SEQ ID NO: 11). FIG. 2 is a digital image of a Western blot. The doses for the two compounds were chosen to produce strong signals on their own. The EPO induced signal is faster than EPO-Rp (5 min, 15 min peak vs 15 min for EPO-Rp, lasting at least 90 min). Thus, the duration of action of EPO-Rp is much longer than with EPO.

FIG. 3: Demonstration of EPO-R stimulation by EPO-Rp. Cells were stimulated for 20 minutes. FIG. 3 is a digital image of a Western blot. Growth hormone receptor-derived peptide (GH
Rp) was used as a control at a concentration of 10 μM that potently enhances growth hormone receptor activity. Active 2.5 U / ml EPO corresponds to approximately 30-100 nM EPO-Rp.

FIG. 4: Dimerization of EPO-Rp is thought to induce intracellular association between JAK2 and EPO-Rp. When an antibody against JAK2 was used as the precipitated antibody, EPO and EPO-Rp were converted to EPO-
R results in an association with JAK2. FIG. 4 is a digital image of a Western blot.

FIG. 5 shows that EPO-Rp selectively activates EPO-receptors. TF1 cells were incubated with 0.1 U / ml EPO, 10 nM TPO, different concentrations of EPO-Rp and TPO or EPO
Stimulation was performed for 20 minutes with the combination of EPO-Rp (at the same concentration). Cells were lysed and immunoprecipitated with anti-phosphotyrosine antibody (PY). Western blot analysis was performed using an anti-EPOR antibody that specifically recognizes EPO-R and an anti-JAK2 antibody that recognizes a kinase that specifically associates with only the activating receptor. FIG. 5 is a digital image of a Western blot. EPO and EPO-R peptides are EPOR (lower panel)
And act synergistically when activating JAK2 (upper panel). TPO does not enhance the signal together with the EPO-R peptide; activation of JAK2 by TPO and EPORp is additive because TPO activates its own receptor that also associates with JAK2. Moreover, the lower panel shows that TPO does not activate EPOR with EPORp, as the signal is due to the presence of the peptide alone.

FIG. 6 shows suppression of EPO-R internalization by EPO-Rp. [ ¹²⁵ ] EPO
Was measured in TF1 cells in the presence or absence of EPO-Rp. Cells are preincubated for 30 minutes at 37 ° C., after which [ ¹²⁵ ] EPO and 15 μl
M peptide concentrate (EPORp) was added. The cells were further incubated at 37 ° C. for different times, and the internalized ligand was measured by the method of acidic washing. The bound ligand was separated from the unbound ligand by spinning the cells in the oil mixture. Ligand is calculated as% of [ ¹²⁵ ] EPO resistant to acidic washes (intracellular) versus total amount of hormone bound to cells. Thus, EPORp suppresses EPO receptor internalization and thus prolongs its cell surface time.

FIG. 7 demonstrates the activation (phosphorylation) of JAK2 kinase by a TPO receptor-derived peptide (TPO-Rp, SEQ ID NO: 10). Different concentrations of peptide (as indicated) were added to the cells for 20 minutes and IP with anti-PY antibody followed by Western blot with anti-JAK2 antibody. This kinase is activated only if it can bind to the dimerized (biologically active) TPO receptor. Therefore, TPORp
Enhances signaling through the TPO receptor.

FIG. 8 shows that TPO-Rp selectively activates the TPO receptor. Cells were stimulated with 0.1 U / ml EPO, 10 nM TPO, different concentrations of TPOR peptide, and a combination of TPO or EPO (at the same concentration) and TPO-R peptide for 20 minutes. Cells were lysed and immunoprecipitated with anti-PY antibody. Western blot analysis was performed by using an anti-EPOR antibody that specifically associates only with the activating receptor (EPOR or TPOR). The figure is a digital image of a Western blot. TPO and TPO-R
Peptides act synergistically when activating JAK2 kinase by TPO-R (top panel). In the same way as activation of JAK2 by EPO and TPORp as an additive, EP
O and TPO-RpEPO do not enhance the signal with the TPO-R peptide. In addition, the signal seen in the lower panel is due to the presence of EPO only,
Do not activate.

FIG. 9 demonstrates that TPORp activates JAK2 kinase via the TPO receptor. The experiment was performed as in FIG. 8, except that the cells were immunoprecipitated using an anti-JAK2 antibody. WB probed with anti-PY antibody (top panel) shows that TPORp activates JAK2 kinase in two ways: it mimics TPO activation and acts synergistically with TPO. WB and anti-JAK2 antibodies (lower panel) indicate that the immunoprecipitates contained the same amount of JAK2 protein.

FIG. 10 shows activation of leptin receptor (OB-R) signaling by leptin (Leptin) receptor-derived peptide (LRp). Transiently transfect COS-1 cells using the long leptin receptor (OB-R ₁ ), 6 nM leptin, 15 μM LRp
Alternatively, stimulation was performed for 20 minutes using a combination thereof. Leptin receptor expression was confirmed by Western blot analysis using specific anti-OBR antibodies and by binding of [ ¹²⁵ ] -leptin to transfected cells. Cells were lysed and immunoprecipitation with anti-JAK2 antibody was performed. WB with the same antibody (left panel) showed that equal amounts of protein were present in all immunoprecipitates. WB using anti-PY antibody
(Right panel) shows that LRp is activated by the leptin receptor on JAK2 because the same enhancement of protein phosphorylation signal was not observed when cells were stimulated with leptin or LRp.
Demonstrate to activate. As shown in the figure, the upper band is phosphorylated JA
The K2 molecule is shown, the central band is a phosphorylated STAT5 molecule (both specific proteins in the OB-R signaling pathway), and the lower band is an unidentified protein whose phosphorylation does not change between unstimulated and stimulated cells. It is. Two additional peptides: 15
Insulin receptor-derived peptide (IRp, SEQ ID NO: 3), a peptide that strongly activates signaling by the insulin receptor at μM concentrations, and also peptide derivatives of the leptin receptor, but with the MHC-1 peptide Cp without any similarity of was used as a specificity control. Each of these peptides
Did not activate JAK2 or STAT5 protein.

FIG. 11 demonstrates that LRp mimics leptin and shows synergy with leptin in activating leptin receptor signaling. COS-1 cells were transfected with the long form of leptin receptor as shown and different concentrations of leptin (OB), LRp and the lowest dose of OB (1 ng / ml) were combined with different concentrations of peptide. And stimulated for 20 minutes. Immunoprecipitation was performed using an anti-JAK2 antibody. Lower panel W
B shows that the immunoprecipitates contain the same amount of JAK2 protein. WB with anti-PY antibody (upper panel) shows activation (phosphorylation) of JAK2 and STAT5 by OB and LRp. Both the natural ligand and the peptide activate JAK2 to the same extent. When combined, there is synergy between them. For example, activation of JAK2 is achieved by adding 1 ng / ml OB and 30 μM LRp together (16 arbitrary units).
More potent than stimulating cells separately with the same compound (2 and 8 units). CP
And Irp, alone or in combination with OB, had no effect on protein phosphorylation. The numbers in the rectangle below the panel indicate the quantification of the Western blot. To do so, the membrane was scanned and the intensity of the protein bands was quantified using the NIH 1.5 image program. The upper value corresponds to the JAK2 protein and the lower value corresponds to STAT5.

FIG. 12 shows the effect of LRp on the activation of STAT5 by leptin receptors.
STAT5 is a transcription factor that specifically binds to activated JAK2, which previously needs to be phosphorylated by interaction with dimerized receptors. The experiment was performed in Example 1.
1, except that an anti-STAT5 antibody was used instead of JAK2. WB with anti-PY antibody (upper panel) shows that LRp mimics the action of leptin (OB) and when combined, it acts synergistically. The lower panel shows that all immunoprecipitates have the same amount of STAT.
5 Indicates that it contains protein.

FIG. 13 demonstrates the effect of LRp on leptin receptor short form (OB-R _S ) signaling. COS-1 cells were transfected with the short form of the leptin receptor and, as shown, different concentrations of leptin (OB), LRp and two different doses of OB (1n
g / ml and 10 ng / ml) were stimulated with different peptide concentrations. IP was performed with an anti-OBR antibody, and then the blot was probed with a JAK2 antibody. The WB in the upper panel indicates that the JAK2 protein associates with the leptin receptor only when the receptor has been activated. LRp indicates that it has the same action as the natural ligand. In the lower panel, cells were IP'd with anti-JAK2 antibody and the membrane was probed with anti-PY. JAK2 phosphorylation (activation) is only observed when both LRp and leptin are present. Thus, LRp and leptin together activate the short form of the leptin receptor (a form of receptor expressed in db / db animals), the effect of which is that if only one of the compounds is added Is not observed.

FIG. 14 demonstrates activation of growth hormone receptor (GHR) by a GHR-derived peptide (GH-Rp, SEQ ID NO: 9). IM9 cells were stimulated with 10 nM GH, 30 μM GHRp and combinations for 20 minutes. IP was performed using an anti-JAK2 antibody. JA
WB with K2 antibody (right panel) shows the same amount of protein present in all immunoprecipitates. WB with anti-PY demonstrates equal activation (phosphorylation) of JAK2 by GH and GHR peptides.

FIG. 15 shows the kinetics for the action of GH and GHRp. The doses for the two compounds are as indicated at the top of the panel. The top panel suggests that, most likely, by inducing a dimeric conformation between the GH-receptors (receptor dimerization would induce an intracellular association between JAK2 and GHR) 2 shows that GH and GHRp phosphorylate JAK2 to the same extent. The GH-inducible signal of JAK2 phosphorylation is GH
Faster than Rp (approximately 30 seconds at 1 minute vs. 5 minutes at least 40 seconds for GHRp) Thus, the duration of action of GHRp is much longer than for GH.

FIG. 16 demonstrates that GHRp (P) extends the range of GH action. IM9
Cells were stimulated with 1 nM, 10 nM and 2 μM GH in the presence or absence of GHRp. These concentrations of GH were used because the GH effect has a bell-shaped dose response curve and shows an IC ₅₀ at a GH concentration of 2 μM. IP was performed using anti-JAK2. WB was performed with the same antibody (right panel) or with anti-PY (left panel). Anti-JAK2
The blots probed with show that the same amount of protein is present in all immunoprecipitates. Anti-PYWB demonstrates that in the presence of 1 nM GH, the peptide enhances JAK2 activation about 3-fold. At a GH inhibitory concentration of 2 μM GHRp, the natural hormonal response was increased about 2-fold. Thus, GHRp extends its range of GH action by increasing its activity at low concentrations and extending its activity to higher hormone concentrations.

FIG. 17 demonstrates the synergistic effect of GHRp on GH action. Cells were stimulated with different concentrations of GHR peptide alone (upper panel) or in combination with 1 nM (middle panel) and 0.1 nM GH (lower panel) for 20 minutes. Implement IP using anti-JAK2 and anti-PY
Bulking B was performed with the antibody. GHRp shows strong synergy with both concentrations of GH:
(I) No effect on JAK2 phosphorylation is observed at low (up to 5 μM) peptide concentrations, but when combined with 1 nM GH, activation of JAK2 is significantly increased (1
: Ii) 0.1 nM concentration of GH has no effect on JAK2 phosphorylation, but in combination with low 0.3 μm GHRp (which also has no effect on itself), JAK2 activation It is increased about four times.

FIG. 18 demonstrates that GHRp enhances STAT5 phosphorylation. STAT5 is GH
Is a molecule downstream of JAK2 in the signaling pathway. The putative mechanism is that the receptor dimerizes (or multiplies) and JAK2 associates with the multimeric receptor and becomes activated by phosphorylation. Next, JAK2 can associate with STAT5. This also becomes associated by phosphorylation. Different concentrations of GHRp as shown at the top of the panel
Alternatively, cells were stimulated with GH and IP was performed with anti-STAT5, followed by WB with anti-PY (top panel) and anti-STAT5 (bottom panel). Increased STAT5 phosphorylation is observed with increasing doses of added GHRp. The same band of phosphorylation was observed when cells were stimulated with GH, indicating specific signaling by GHR. The lower panel shows that all immunoprecipitates contained the same amount of STAT5 protein.

FIG. 19 demonstrates that GHRp, together with GH, synergizes STAT5 activation (phosphorylation). Stimulation of cells with 0.2 nM GH in the presence or absence of GHRp shows a significant increase in STAT5 phosphorylation, e.g., phosphorylation is 0.2 nM GH or 1.
25 μM GHRp alone is not observed, but the signal is increased when both compounds are present. Furthermore, the weak signal of STAT5 protein phosphorylation by 2.5 μM peptide is strongly enhanced by the presence of 0.2 nM GH. Activity was measured using an anti-PY antibody (upper panel). The lower panel shows that the same amount of STAT5 is present in all immunoprecipitates. Thus, GHRp is measured by STAT5 phosphorylation,
It has a very strong synergistic effect on GH.

FIG. 20 shows the selectivity of GHRp for the GH receptor. GHRp, IGF-IRP (a peptide selected and active on IGF-1R, SEQ ID NO: 5) and P1-Rp (a peptide specific for the prolactin receptor, SEQ ID NO: 35; prolactin receptor is high on GHR [ ¹²⁵ ] GH internalization was measured in IM9 cells in the presence of levels (with about 50% identity). Cells were pre-incubated for 30 minutes at 37 ° C., after which [ ¹²⁵ I] GH and different concentrations of peptide were added as indicated. At 37 ° C
After incubation for 20 minutes, the internalized ligand was determined by the method of acidic washing. The cells were spun in the oil mixture to separate the bound ligand from the unbound ligand. Internalized ligand is calculated as% of [ ¹²⁵ I] -GH resistant to acidic washes (intracellular) versus total amount of hormone bound to cells. Thus, GHRp selectively inhibits GH-receptor internalization and thus prolongs the cell surface time of the receptor.

[Procedure amendment]

[Submission date] September 19, 2000 (2000.9.19)

[Procedure amendment 2]

[Document name to be amended] Drawing

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[Document name to be amended] Abstract

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Abstract: An oligopeptide having an amino acid sequence corresponding to the extracellular domain of a receptor and having a sequence similar to a regulatory peptide from an MHC class I antigen is capable of exhibiting a physiological response of a ligand binding to the corresponding receptor. Raise or replace this. The oligopeptides are used in the diagnosis and treatment of diseases involving inadequate or inappropriate receptor responses, and in the screening of drug candidates that affect receptor surface expression. If the sequence corresponding to the regulatory peptide is modified or deleted, the modified receptor molecule is also useful for drug screening.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 C07K 14/705 33/50 C12N 15/00 ZNAA // C07K 14/705 A61K 37/02 ( 81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C , CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (29)

[Claims] 1. A method for regulating the activity of a type 2 cell surface receptor containing an activating sequence, comprising binding an exogenous compound to the activating sequence. 2. The method of claim 1, wherein said binding is in the absence of any exogenous ligand that normally activates said cell surface receptor. 3. The binding is usually made in the presence of a ligand that activates the receptor, and when the level of activation uses a combination of a ligand and an exogenous compound, The method of claim 1, wherein the method is higher than with the same amount of ligand alone. 4. The method of claim 1, wherein said modulation is an increase in activity. 5. The method of claim 1, wherein said modulating is a reduction in activity. 6. The cell surface receptor is an insulin-responsive glucose transporter, a leptin receptor, a low density lipoprotein receptor, a granulocyte colony stimulating factor receptor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12
Interleukin receptor, including IL-13, IL-15 and IL-17 receptors, human growth hormone receptor, VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, transferrin receptor, prolactin receptor 2. The method of claim 1, wherein the method is selected from the group consisting of: a T cell receptor; a CNF receptor; 7. The exogenous compound has at least about 8 amino acids and about 40 amino acids having an amino acid sequence corresponding to the activation sequence of the extracellular domain of a cell surface receptor.
2. The method of claim 1, wherein the method is an oligopeptide comprising less than three amino acids. 8. The oligopeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2
7, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32
The method of claim 7, wherein the method is selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. 9. The oligopeptide of any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2
7, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32
The method of claim 7, wherein the method is at least about 90% homologous to a sequence selected from the group consisting of: SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35. 10. The method of claim 1, wherein said activation comprises a configuration change sufficient to elicit a phosphorylation event. 11. The method of claim 1, wherein said cell surface receptor is on the surface of a cell. 12. The method of claim 11, wherein said cells are in vivo. 13. The method according to claim 11, wherein said cells are human cells. 14. A method for modulating the activity of a type 2 cell surface receptor containing an activating sequence on the surface of a mammalian cell, said method comprising binding an exogenous compound to said activating sequence. . 15. The method of claim 14, wherein said binding is normally in the absence of any exogenous ligand that activates said cell surface receptor. 16. The method according to claim 16, wherein said binding is usually in the presence of a ligand which activates said receptor, and wherein said level of activation is with a combination of ligand and exogenous compound. Is higher than when using the same amount of ligand alone. 17. A method of treating a patient having a disease normally associated with a ligand that activates a type 2 cell surface receptor, comprising administering an exogenous compound that binds to the receptor activation sequence. The above method, comprising: 18. The method of claim 1, wherein said binding is made in the absence of an exogenous ligand.
7. The method according to 7. 19. The binding is made in the presence of an exogenous ligand and the level of activation is the same when the combination of ligand and exogenous compound is used. 18. The method of claim 17, wherein the method is higher than the child alone. 20. Adding a candidate bioactive agent to a target comprising an activation sequence for a cell surface receptor and determining whether said candidate bioactive agent is bound to said activation sequence. A screening method comprising: 21. The method of claim 20, wherein said target comprises a full-length cell surface receptor. 22. The method of claim 20, wherein said target is labeled. 23. The candidate bioactive agent is labeled.
The method described in. 24. The following steps: a) At least about 8 amino acids and less than about 40 amino acids having an amino acid sequence corresponding to the activation sequence of the extracellular domain of a cell surface receptor and a type 2 cell surface receptor. An oligopeptide that binds to the activating sequence of the cell surface receptor, b) adding a candidate bioactive agent, and c) the candidate bioactivity to the activating sequence. A screening method, comprising: determining the binding of an agent. 25. A screening method for a biologically active agent capable of regulating the activation of a type 2 cell surface receptor, comprising the steps of: converting a candidate biologically active agent to a type 2 cell surface receptor containing an activating sequence; The screening method comprising the steps of: adding to a mammalian cell to be included; and determining whether or not the candidate bioactive agent binds to the activating sequence to regulate the activation of the receptor. 26. The method of claim 25, wherein said modulation is an increase in activity. 27. The method of claim 25, wherein said modulating is a reduction in activity. 28. The method of claim 25, wherein said oligopeptide is labeled. 29. The candidate bioactive agent is labeled.
The method described in.