Compounds and peptides that bind to the erythropoietin receptor (EPO-R)

专利摘要:
The present invention binds to EPO-R or acts as an EPO agonist and consists of amino acid residues of 10 to 40 or more in length, the sequence of which is X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO) And peptides thereof, wherein each amino acid has the following standard abbreviation: X 3 is C; X 4 is R, H, L, or W; X 5 is M, or F; X 6 is each selected from any of twenty genetically encoded L-amino acids or stereoisomer D-amino acids; X 7 is D, E, I, L, or V; X 8 is C.
公开号:KR19990022656A
申请号:KR1019970709231
申请日:1996-06-07
公开日:1999-03-25
发明作者:씨. 라이튼 니콜라스；제이. 도워 윌리엄；에스. 창 레이；케이. 케쉬얍 아런；케이. 졸리프 린다；존슨 다나；멀케히 린다
申请人:린골드 고돈；아피맥스 테크놀로지스, 엔.브이.；존 더블유. 하버；오르토 파마슈티칼 코오포레이숀；
IPC主号:

专利说明:

Compounds and peptides that bind to the erythropoietin receptor (EPO-R)
EPO is a glycoprotein hormone with a molecular weight of about 34,000 with 165 amino acids, four glycosylation sites on positions 24, 38, 83 and 126 of the amino acids. EPO is initially produced as a precursor protein with a single peptide consisting of 23 amino acids. EPO comes in three forms: alpha, beta, and asialo. The alpha and beta forms have slightly different carbohydrate components, but have the same efficacy, the same biological activity, and the same molecular weight. Asianrotypes are alpha and beta forms with terminal carbohydrates (cyrianic acid) removed. DNA sequences encoding EPO have been reported in US Pat. No. 4,703,008 (Lin, 1987).
EPO is a substance that stimulates mitosis and diversification of erythrocyte progenitor cells to produce erythrocytes, which are produced in the kidney when hypoxia predominates. During the diversification of erythroid progenitor cells induced by EPO, globin synthesis is induced and the synthesis of ham complexes and the number of ferritin receptors increase. This not only allows the cells to produce higher amounts of iron, but also the synthesis of functional hemoglobin. Since hemoglobin in mature erythrocytes binds to oxygen, erythrocytes and hemoglobin contained in them play an important role in supplying oxygen to the body. The process of formation of the complex as disclosed is caused by the interaction of EPO with appropriate receptors on the cell surface of erythroid progenitor cells (see Graber and Krantz (1978) Ann. Rev. Med. 29: 51-66).
When the body is in a healthy state, MEPO is present at low concentrations in plasma because tissue is supplied with sufficient oxygen from the number of red blood cells present. This low concentration of steady state is sufficient to stimulate the replacement of red blood cells, which are usually lost due to aging.
The amount of EPO in circulation increases under hypoxia conditions when the amount of oxygen carried by blood cells in circulation decreases. Hypoxia can be caused by massive blood loss through bleeding, destruction of red blood cells due to overexposure to radiation, decreased oxygen intake from high altitudes or unconsciousness over long periods of time, or various forms of anemia. have. EPO increases erythrocyte production by stimulating the proliferation of erythrocyte progenitor cells in response to tissues undergoing hypoxic tension. The amount of EPO decreases when the number of red blood cells in circulation is greater than normal tissue oxygen demand.
Since the EPO hormone is essential for the process of red blood cell formation, the hormone has ample potential for its useful use in the diagnosis and treatment of blood diseases due to the production of low or deficient red blood cells. Recent research has been based on the topic of EPO's efficacy in treating various disease states, disorders, and hematologic lesions. Examples of these include: Beta thalassemia (also referred to as thalassemia, Vedovato et al., 1984, Acta. Haematol. 71: 211-213); Cystic fibrosis (see Vichinsky et al., 1984, J. Pediatric 105: 15-21); Pregnancy and mental illness (see Cotes et al. 1983, Brit. J. Ostet. Gyneacol. 90: 304-311); Early anemia due to immaturity (see Haga et al., 1983, Acta Pediatr. Scand. 72: 827-831); Spinal cord injury (see Claus-Walker et al., 1984, Arch. Phys. Med. Rehabil. 65: 370-374); Spatial escape (see Dunn et al., 1984, Eur. J. Appl. Physiol. 52: 178-182); Acute blood loss (see Miller et al., 1982, Brit. J. Haematol. 52: 545-590); Aging (see Udupa et al., 1984, J. Lab. Clin. Med. 103: 574-580 and 581-588, Lispschitz et al., 11983, Blood 63: 502-509); Various tumorigenic diseases accompanied by abnormal red blood cell production (see Dainiak et al., 1983, Cancer 5: 1101-1106 and Schwartz et al., 1983, Otolaryngol, 109: 269-272); And renal failure (Eschbach et al., 1987, N. Eng. J. Med. 316: 73-78).
Hewick's US Pat. No. 4,677,195 describes purified and uniform EPO. DNA sequences encoding EPO were purified and cloned and expressed to produce synthetic polypeptides having the same biochemical and immunological properties. Recombinant EPO molecules have been produced that have the same oligosaccharides as oligosaccharides on natural materials (Sasaki et al., 1987, J. Biol. Chem. 262: 12059-12076).
Despite the usefulness of purified recombinant EPO, little is known about the mechanism of proliferation and diversity of erythrocytes induced by EPO. The specific interaction of EPO with progenitor cells of immature red blood cells, platelets, and megakaryocytes still needs to be elucidated. The reason for the lack of elucidation is at least in part due to the low number of surface EPO receptor molecules on normal erythroblasts and erythroleukemic cell lines. This is shown in the literature: Krantz and Goldwaser (1984) Proc. Natl. Acad. Sci. USA Letters 211: 229-233; Mufson and Gesner (1987) Blood 69: 1485-1490; Sakaguchi et al. (1987) Biochem. Biophys. Res. Commun. 146: 7-12; Sawyer et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3690-3694; Sawyer et al. (1987) J. Biol. Chem. 262: 5554-5562; and Todokoro et al. (1988) Proc. Natl. Acad. Sci. USA 84: 4126-4130.
The crosslinked complex of radial iodinated EPO with cell surface protein suggests that the cell surface protein contains two polypeptides, each having about 85,000 Daltons and 100,000 Daltons, respectively. In particular, the two crosslinked complexes were found to have the same peptide fragment undergoing V8 protease digestion, suggesting that the two EPO binding polypeptides may be products of the same or very similar genes (see Sawyer et al., 1988, supra). ). However, most cell surface binding studies have shown a single class of binding sites consisting of 300-600 for one cell surface, with a Kd of about 800 pM (pico molarity) (Sawyer et al., 1987, Proc. Natl. Acad. Sci. USA 84: 3690-3694). However, erythrocytes of EPO-reactive spleen from rats injected with the Anemia strain of Friend Leukemia virus have been shown to have high affinity and low affinity, respectively, with dissociation constants of 100 pM and 800 pM, respectively. (See Sawyer et al., 1987, J. Biol. Chem. 262: 5554-5562 and Landschulz, 1989, Blood 73: 1476-1478). DNA sequences and encoded peptide sequences of mouse and human EPO receptor proteins have been disclosed (D'Andrea et al., PCT Patent Publication No. WO 90/08822, published in 1990).
The cloned genes of EPO-R facilitate the study of agonists and antagonists of this important receptor. The utility of recombinant receptor proteins enables the study of receptor-ligand interactions in random and semi-random peptide diversity generation systems. These systems include peptides on the plasmids described in US patent application 778,233, filed Oct. 16, 1991, and phage peptides of US Pat. App. No. 718,577, filed Jun. 20, 1991; Proc., Published in August 1990 by Cwirla et al. Natl. Acad. Sci. USA 87: 6378-6382; Encrypted synthetic library (ESL) system of US Patent Application No. 946,239, filed September 16, 1992; Large scale immobilized polymer synthesis systems described in US Patent Application No. 492,462, filed March 7, 1990; PCT Patent Application Publication No. 90/15070, published December 13, 1990; Fodor et al. 15 February 1991, Science 251: 767-773; Dower Fodor, 1991, Ann. Rep. Med. Chem. 26: 271-180; US Patent Application No. 805,727, filed December 6, 1991.
However, there is still an urgent need for compounds that bind to or otherwise interact with EPO-R in two cases: the study of important biological activities mediated by this receptor and the treatment of diseases. The present invention provides such compounds.
This application is directed to US patent applications Ser. No. 08 / 484,635 and 08 / 484,631, filed Jun. 7, 1995, which is a CIP application of US patent application Ser. No. 08 / 155,940, filed Nov. 19, 1993, which is incorporated herein by reference. CIP application.
The present invention relates to peptides and compounds that bind to and activate erythropoietin receptors (EPO-R) or otherwise function as EPO agonists. The present invention is in the field of biochemistry and medical chemistry, and provides the EPO agonists for the treatment of diseases in humans in particular.
1 depicts the sequences of the mutant oligomers used in the procedures described herein.
2-1 and 2-2 show exemplary peptide sequences of the present invention.
3 shows a novel phagemid variant library using the pVIII display system. Two cysteines as well as tyrosine, glycine-proline and threonine-tryptophan residues (underlined) were fixed in this library. Other positions between the cysteine residues (represented by the underlined italics present in the original AF11154 heat peptide) can be mutated by the oligostructure to change each amino acid residue at 50% frequency. X represents a random amino acid position.
4A-4C depict the pIII and pVIII phagemid variant libraries currently constructed and screened. Amino acid residues in bold typeface are fixed and the remainder (denoted by NNK) is randomized. 4A (ON3007) and 4C (ON3017) were designed to study the contribution of additional adjacent regions, the N-terminus and C-terminus, respectively, to the core sequence (second cysteine in tyrosine). 4B (ON3016) has a peptide Y-CHFGPLTWVC backbone, where the tyrosine residue is located immediately after the first cysteine. This library also has additional optional residues on either side of the core sequence.
FIG. 5 shows a variant library, GGTYSCHFGPLTWVCKPQGG, constructed and screened using the novel Lac-I display vector (headpiece dimer). The amino acid residues represented by X are random (NNK), the underlined ones are slightly mutated (91: 3: 3: 3/91: 3: 3: 3 / K), while the outlined ones are highly mutated. (70: 10: 10/70: 10: 10 / K).
FIG. 6 shows the TIAQYICYMGPETWECRPSPKA variant library as constructed and screened using the novel Lac-I display vector (headpiece dimer). The amino acid residues represented by X are random (NNK), the outlined ones are fixed, and the two glutamic acid residues are slightly mutated (91: 3: 3: / 91: 3: 3: / K).
FIG. 7 graphically depicts FDCP-1 / hEPO-R biological assay results for selection from the following peptides:
● shows the result of GGCRIGPITWVCGG,
(Circle) shows the result of GGLYLCRFGPVTWDCGYKGG,
■ shows the result of GGTYSCHFGPLTWVCKPQGG,
▲ shows the result of GGDYHCRMGPLTWVCKPLGG,
▼ shows the results of VGNYMCHFGPITWVCRPGGG,
| Shows the result of GGVYACRMGPITWVCSPLGG,
+ Represents the result of VGNYMAHMGPITWVCRPGG,
* Indicates the result of EPO.
FIG. 8 graphically shows the results of TF-1 bioassay of EPO (▲ and ◆ show assay results using 10,000-20,000 cells for EPO and one well); In addition, the graph shows the results of biological analysis of the peptide VGNYMCHFGPITWVCRPGGG (SEQ ID NO :) (■ and ● shows the analysis results using 10,000-20,000 cells for the peptide and one well).
9A (control), FIG. 9B (treated with 500 pM EPO) and FIG. 9C (treated with 25 mm GGDYHCRMGPLTWVCKPLGG) show MTT proliferation assay results at 200 × magnification for representative colonies of spleen cells of mice treated with phenylhydrazine. It was taken.
FIG. 10 is a graph graphically showing the results of FDCP-1 / hEPOR bioassay showing improved efficacy of biotinylated peptide (ie GGTYSCHFGPLTWVCKPQGGSSK), Ahx-biotin by performing oligomerization with streptavidin. Peptides GGTYSCHFGPLTWVCKPQGG and GGTYSCHFGPLTWVCKPQGGSSK (Ahx-Biotin) -streptavidin complexes exhibit approximately the same activity, but when the latter peptide is precomplexed with streptavidin, the efficacy is greater. In this assay, activity appears to be rarely reduced at the lowest concentration (5 nM for peptide in the complex). Streptavidin alone has the lowest activity at high concentrations.
11 graphically depicts the results of FDCP-1 / hEPOR indicating that the efficacy of peptide AF11505 was increased in polyclonal goat anti-biotin-modulated. Precomplexes of GGTYSCHFGPLTWVCKPQGGSSK (Ahx-biotin) and goat's anti-biotin antibody (GGTYSCHFGPLTWVCKPQGGSSK + G anti-B) show that the efficacy of one log of peptides on the free peptide was increased. Purified antibody (G anti-B) did not have a stimulating effect.
Figure 12 shows a synthetic scheme for the dimeric peptide analog preparation of GGTYSCHFGPLTWVCKPQGG.
Figure 13 depicts IC ₅₀ of dimeric peptide analogs of GGTYSCHFGPLTWVCKPQGG. Affinity was measured using a radioligand competition binding assay for EPO-R with PIG label immobilized on mAB179.
FIG. 14 depicts in vitro biological activity of dimeric peptide analogs of GGTYSCHFGPLTWVCKPQGG using FDCP-1 / hEPOR cell proliferation assay. Dimeric peptides have an EC ₅₀ of about 20 nM with a 20-fold increase in potency over the parent peptide.
15 shows the cell cycle progression of FDC-P1 / ER.
FIG. 16 shows an equilibrium binding assay for specific binding of EBP and [ ¹²⁵ I] EPO immobilized on agarose beads, showing 5 nM ± 2Kd based on linear Scatchard of binding isotherms.
17A, 17B and 17C show the results of biological analysis of hypoxia-reduced hypoxia mice using GGTYSCHFGPLTWVCKPQGG, GGTYSCHFGPLTWVCKPQ and LGRKYSCHFGPLTWVCQPAKKD, respectively.
18A and 18B show the results of a biological analysis of mice with reduced erythrocyte-rich hypoxia using GGTYSCHFGPLTWVCKPQGG (AF12080) dimeric peptide analogue containing peptide TIAQYICYMGPETWECRPSPKA and two disulfide bonds.
19A and 19B show the results of reticulocyte analysis using EPO and GGTYSCHFGPLTWVCKPQGG. Ten animals were used in two dose groups. EPO total dose; 0.4, 4.0, 40.0 units / mouse, total dose medium control; GGTYSCHFGPLTWVCKPQGG; 2.0, 20.0, 200.0 μg / mouse, total dosage medium control + DMSO (1-2%) 200 μg / mouse = 10 mg / kg Ratio: In vitro proliferation assay data has the following ratios; 2 μg of GGTYSCHFGPLTWVCKPQGG = 0.4 units of EPO.
20A and 20B show the results of reticulocyte analysis using EPO and GGTYSCHFGPLTWVCKPQGG, respectively. Ten groups of two groups of sires were used. EPO total dose: 0.4, 4.0, 40.0 units / mouse; GGTYSCHFGPLTWVCKPQGG: Total dose when increased to an amount of 1 mg / mouse = 50 mg / kg, and total dose when increased to an amount of 2 mg / mouse is mg / kg.
Preferred Embodiments of the Invention
The following terms have the following common meanings;
Amino acid residues in peptides have the following abbreviations: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; Proline is Pro or P; threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Asphaltic acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; tryptophan is Trp or W; Arginine is Arg or R; And glycine is Gly or G.
Suitable components of the compounds of the present invention include stereoisomers of twenty conventional amino acids (e.g. D-amino acids), non-natural amino acids such as a, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, other unknown amino acids, and the like. It is possible. Examples of unknown amino acids here include 4-hydroxyproline, O-phosphoseline, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and other similar amino acids and imino acids (eg 4-hydroxyproline).
An agonist refers to a biologically active ligand, which binds to its complementary biologically active receptor, causing a biological reaction in the latter or enhancing the biological activity of the receptor already.
Host cells refer to prokaryotic or eukaryotic cells or groups of cells that can be transformed or transformed by recombinant DNA vectors. For the purposes of the present invention, prokaryotic cells are preferred.
Peptides or polypeptides refer to polymers of alpha amino acids in which monomers are linked together through amide bonds. Peptide refers to two or more amino acid monomers.
Non-toxically acceptable alkali metal salts, alkaline earth metal salts, and ammonium salts are commonly used in the pharmaceutical arts, and examples of these include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts. Which are prepared according to methods known in the art. Pharmaceutically acceptable salts also include nontoxic acid addition salts, which are prepared by reacting the compounds of the present invention with suitable organic or inorganic acids. Examples of representative salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, tosylate, acetate, oxalate, valate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, horses Raises, fumarates, succinates, tartrates, lead silates, and the like.
A pharmaceutically or therapeutically effective amount or dosage refers to an amount sufficient to induce a desired biological result. Biological outcome herein refers to alleviation of the symptoms, signs, or causes of a disease or modification of a biological system to any other desired form. Preferably this dose or dose is sufficient to stimulate EPO-R, thereby alleviating symptoms associated with deficiency of EPO or associated with defective or low red blood cell counts in vivo.
Recombinant DNA cloning or expression vectors can be used to transform host cells and refer to DNA or RNA molecules that encode useful functions. For the purposes of the present invention, cloning vectors generally serve as intermediates in the construction of expression vectors; The latter vector is used to plant or transform host cells so that the transduced host cells produce a protein or other product encoded by the vector. These vectors are commonly referred to as plasmids, which are vectors that can be extrachromosomal in the host cell but can also be integrated into the genome of the host cell. Those skilled in the art call this cloning vector, vector, expression vector, plasmid.
The present invention provides compounds that bind to and activate EPO-R or otherwise act as EPO agonists. These compounds include lead peptide compounds found by any peptide diversity generation system, and derivative compounds. This derivative compound has the same or similar molecular structure or morphology as the lead compound but is sensitive to hydrolysis or proteolysis and / or other biological properties such as increased affinity of the receptor, increased activity in vivo and in vitro And lead compounds.
The various systems for generating random peptides initially used included the phage peptides described above. Random peptides have amino acid residues of length 8, with Cys residues adjacent to both ends (thus the total length is 10 amino acids). In these early systems, random peptides have been shown as part of a fusion protein containing the pVIII coat protein of the phage fd derivative (peptide on phage). Along with the DNA encoding this fusion protein, the fusion protein is panned onto immobilized EPO-R. The screening process involves incubating the fusion protein and the immobilized receptor multiple times to collect the fusion protein (with accompanying DNA) bound to the receptor and to produce a large number of the fusion protein.
In general, after three screening operations, the fusion protein and the accompanying DNA are isolated and cultured to produce a fusion protein preparation for ELISA analysis, and then determine whether the fusion protein specifically binds to the receptor. The assay is performed similar to the screening procedure, except that after removing the unbound fusion protein, the wells are treated with rabbit antiphage antibodies (or anti-lac antibodies for peptides on the plasmid system), followed by the analysis of alkaline phosphatase in each well. The amount is different from the standard method. Comparing test wells with control wells (without receptors) can determine whether the fusion protein is specifically bound to the receptor.
Immobilized receptors used for selection and ELISA analysis consisted of the extracellular domain of EPO-R and were produced in recombinant host cells. These receptor molecules can be produced in a variety of different forms and host cells. One useful form is a form made of a signal peptide for protein secretion and binding attachment of a glycophospholipid membrane (this attachment form is the so-called PIG-tailing as Caras and Weddell, March 3, 1987, Science 243: 1196-1198 and in Lin et al., Aug. 10, 1990, Science 249; 677-679). This system allows the receptor to degrade from the surface of the cell expressing the receptor and helps the degraded receptor to collect fairly well. Preferably the protease cleavage site, eg the thrombin site, is interposed between the PIG labeled receptor and the receptor itself.
The recombinant receptor protein was immobilized using the following method. Microtiter plates were coated with antireceptor antibodies and the wells containing the immobilized receptors were treated with bovine serum albumin (BSA) to block nonspecific binding. PIG-labeled receptors with thrombin degradation sites were added to coated wells of microtiter plates and subsequently washed to remove unbound receptors.
When using a random peptide generation system that allows for ligand-receptor interaction of polyatoms, it should be noted that the density of immobilized receptors is an important factor in determining the affinity of ligands to bind immobilized receptors. At higher receptor densities (ie, when each antireceptor antibody coated well is treated with 0.25 to 0.5 mg of receptor), multivalent binding is less than at lower receptor densities (ie each antireceptor antibody coated well is 0.5-1 ng May be better treated if When multivalent bonds occur, it is easier to separate ligands with relatively low affinity. In general, immobilized high density receptors can be used to identify lead compounds, and then derivatives of lead compounds were tested under low receptor densities to separate compounds with higher affinity of the receptor than lead compounds.
Often, receptors were added only to alternating rows of microtiter plates; BSA-blocked wells in the blank row were used as negative controls to determine whether receptor-specific responses resulted in the observed results. The fusion protein preparation is then added to the wells and incubated before binding to the receptor occurs; The wells were then washed to remove unbound fusion protein.
Using the system described above, a peptide was found that binds to EPO-R. The DNA encoding this fusion protein bound to the receptor was sequenced. This peptide had the following sequence: GCCRIGPITWVCGG (SEQ ID NO :) (N-terminal GG residue allows degradation on phage). The peptide showed low affinity for BSA and anti-receptor antibodies as measured by ELISA, while high affinity for EPO-R. Phagemid clones also competed with free EPO and cognate free peptides. Moreover, free peptides have been found to compete with EPO-phagemid, LacI-EPO fusions, and radioligands. Finally, the free peptides did not compete in the IL-2Rαβ binding assay.
Mutation studies were performed on these preferred peptides. To collect oligonucleotides encoding random peptides, the mutant oligomers of FIG. 1 were prepared where N is nucleotides A, C, G, or T (molar amounts; depending on the method used, other nucleotides may be used) K was G or T (equivalent) in the codon motif (NNK). Those skilled in the art will appreciate that such NNK motifs encode all amino acids, encode only one stop codon, and reduce codon bias. There are 32 possible codons derived from the NNK motif; One is a codon for 12 amino acids each, two are codons each for 5 amino acids, 3 are codons each for 3 amino acids, and only one exists for the 3 stop codons.
Mutant fusion proteins and DNA encoding them were reselected on immobilized EPO-R. The fusion protein and the accompanying DNA are isolated and cultured to generate a fusion protein preparation and used for ELISA analysis to determine whether the fusion protein specifically binds to the receptor. Preferred peptides in this study are shown in FIG. 2. These peptides are characterized by the motif X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ (SEQ ID NO :), wherein each amino acid denoted by an abbreviation has the following meaning; X ₆ is each selected from one of 20 genetically encoded L-amino acids or D-amino acids, which are stereoisomers; X ₃ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc, where Hoc is homocysteine; X ₄ may be R, H, L or W; X ₅ is M, F, or I; X ₇ may be D, E, I, L, or V; X ₈ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc when X ₃ or X ₈ is one of C or Hoc, where Hoc is homocysteine.
The phage library was screened using an affinity selection protocol to ensure that the affinity of the peptide was approximately even. Here the peptide competed with EPO (100 nM). This process was repeated with two screening operations. The peptide with high affinity was selected by calibrating the temperature of the wash solution (ambient temperature at 4 ° C.) as well as the competitive temperature (ambient temperature at 4 ° C.) and time (15-30 min) during the last two screening runs. Using this affinity selection procedure, peptides with the usual motif X ₁ YX ₂ CX ₄ X ₅ GPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO) were isolated:
Wherein X ₁ , X ₂ , X ₆ , X ₉ , X ₁₀ , X ₁₁ are each selected from 20 genetically encoded L-amino acids or stereoisomer D-amino acids; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V. In a more preferred embodiment, the peptide comprises the amino acid sequence X ₁ YX ₂ CX ₄ X ₅ XGPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO), wherein X ₄ is R or H; X ₅ is F or M; X ₆ is I, L, T, M or V; X ₇ is D or V; X ₉ is G, K, L, Q, R, S or T; X ₁₀ is A, G, P, R or Y. In a most preferred embodiment, the core sequence is the amino acid sequence of X ₁ YX ₂ CX ₄ X ₅ GPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO: ₁₁ ), wherein X ₁ is D, E, L, N, S, T Or V; X ₂ is R or H; X ₂ is A, H, K, Lm M, S or T; X ₉ is K, R, S or T; X ₁₀ is P.
Examples of representative peptides belonging to this motif and isolated in the process of affinity selection are shown in the following table;
Peptides from Affinity Selected Protocals 100 nM EPO, 15 minutes, competitive at 4 ° C GGWVTCRMGPITWVCGVHGG (SEQ ID NO :) GGQLLCGIGPITWVCRWVGG (SEQ ID NO :) GGLYLCRMGPVTWECQPRGG (SEQ ID NO :) GGKYSCFMGPTTWVCSPVGRGV (SEQ ID NO :) GGWVYCRIGPITWVCDTNGG (SEQ ID NO :) GGIYKCLMGPLTWVCTPDGG (SEQ ID NO :) GGMYYCRMGPMTWVCKGAGG (SEQ ID NO :) No competition GGTTQCWIGPITWVCRARGG (SEQ ID NO :) GGPYHCRMGPITWVCGPVGG (SEQ ID NO :) GGEYLCRMGPITWVCERYGG (SEQ ID NO :) GGEYRCRMGPISWVCSPQGG (SEQ ID NO :) GGNYTCRFGPLTWECTPQGGGA (SEQ ID NO :) GGNYVCRMGPITWICTPAGG (SEQ ID NO :)
Peptides from Affinity Selected Protocals 100 nM EPO, 15 minutes, competitive at 4 ° C GGSWDCRIGPITWVCKWSGG (SEQ ID NO :) GGDYTCRMGPMTWICTATRG (SEQ ID NO :) GGDYNCRFGPLTWVCKPSGG (SEQ ID NO :) GGSYLCRMGPTTWLCTAQRG (SEQ ID NO :) GGDYHCRMGPLTWVCKPLGG (SEQ ID NO :) VGNYMCHFGPITWVCRPGGG (SEQ ID NO :) GGLYLCRMGPQTWMCQPGGG (SEQ ID NO :) GGTYSCHFGPLTWVCKPQGG (SEQ ID NO :) GGDYVCRMGPMTWVCAPYGR (SEQ ID NO :) GGLYECRMGPMTWVCRPGGG (SEQ ID NO :) GGWYSCLMGPMTWVCKAHRG (SEQ ID NO :) GGGKYYCWMGPMTWVCSPAGG (SEQ ID NO :) No competition GGYVMCRIGPITWVCDIPGG (SEQ ID NO :) GSCLQCCIGPITWVCRHAGG (SEQ ID NO :) GGNYFCRMGPITWVCQRSVG (SEQ ID NO :) GGEYICRMGPLTWECKRTGG (SEQ ID NO :) GGLYACRMGPITWVCKYMAG (SEQ ID NO :) GGQYLCTFGPITWLCRGAGGGS (SEQ ID NO :) GGVYACRMGPITWVCSPLGG (SEQ ID NO :) GGYTTCRMGPITWVCSAHGG (SEQ ID NO :)
Peptides from Affinity Selection Protocals 100 nM EPO, 15 minutes, competitive at room temperature VGNYMCHFGPITWVCRPGGG (SEQ ID NO :) GGTYKCWMGPMTWVCRPVGG (SEQ ID NO :) GGTYSCHFGPLTWVCKPQGG (SEQ ID NO :) GGDYHCRMGPLTWVCKPLGG (SEQ ID NO :) GGNYYCRFGPITFECHPTGG (SEQ ID NO :) GGLYACHMGPMTWVCQPLRGGGS (SEQ ID NO :) GGEYKCYMGPITWVCKPEGG (SEQ ID NO :) GGDYVCRMGPMTWVCAPYGRGGS (SEQ ID NO :) No competition GGNYVCRMGPITWICTPAGG (SEQ ID NO :) GGDYTCRMGPMTWICTATRG (SEQ ID NO :) GGEYLCRMGPMTWVCTPVGG (SEQ ID NO :) GGSYLCRMGPTTWLCTAQRGGGN (SEQ ID NO :) GGLYTCRMGPITWVCLPAGG (SEQ ID NO :) GGLYKCRMGPMTWVCSPFGG (SEQ ID NO :)
Peptides from Affinity Selection Protocals 100 nM EPO, 30 minutes, at room temperature GGTYSCHFGPLTWVCKPQGG (SEQ ID NO :) GGDYHCRMGPLTWVCKPLGG (SEQ ID NO :) VGNYMCHFGPITWVCRPGGG (SEQ ID NO :) No competition GGDYVCRMGPMTWVCAPYGR (SEQ ID NO :) GGDYNCRFGPLTWVCKPSGG (SEQ ID NO :)
A variant library of 6 amino acid residues in which two cysteine residues were adjacent to adjacent random 8-mers was screened for immobilized EPO-R. Mutant fusion proteins and DNA encoding them were selected on immobilized EPO-R. The fusion protein and the accompanying DNA were isolated and cultured to generate a fusion protein preparation, which was then used for ELISA analysis to determine whether the fusion protein specifically bound to the receptor. Preferred peptides in this study are represented by the sequence GGPHHVYACRMGPLTWIC (SEQ ID NO :), which is therefore surrounded by the core sequence YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ (SEQ ID NO) where each amino acid denoted by abbreviation Has the following meaning; X ₂ and X ₆ are each selected from one of 20 genetically encoded L-amino acids or D-amino acids which are stereoisomers; X ₃ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc, where Hoc is homocysteine; X ₄ may be R, H, L or W; X ₅ is M, F, or I; X ₇ may be D, E, I, L, or V; X ₈ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc when X ₃ or X ₈ is one of C, or Hoc, where Hoc is homocysteine. Wherein the core sequence is coupled to six amino acid units at its amino terminus and each amino acid is each selected from one of the genetically encoded L-amino acids or the stereoisomer D-amino acids.
IC ₅₀ was calculated for some of the peptides belonging to the generic motif described above. These values were measured using free peptides and the results are shown in Table 5 below, where each peptide is individually synthesized and C-terminally amidated, but is readily available as free carboxylic acid or ester or other carboxy amide. Can be manufactured.
Peptide IC ₅₀GGLYLCRFGPVTWDCGYKGG (SEQ ID NO :) 1.67 μM GGTYSCHFGPLTWVCKPQGG (SEQ ID NO :) 237 nm GGDYHCRMGPLTWVCKPLGG (SEQ ID NO :) 179 nm VGNYMCHFGPITWVCRPGGG (SEQ ID NO :) 420nm GGVYACRMGPITWVCSPLGG (SEQ ID NO :) 352 nm VGNYMAHMGPITWVCRPGG (SEQ ID NO :) 67 μM
In addition to the foregoing, other variations of the high affinity peptide EPO agonist of the peptide under conditions concentrating the high affinity peptide have been conducted. As described above, the library was screened using an affinity selection protocol to enrich peptides with high affinity. In this case the peptide competes with EPO (100 nM). This process is repeated as two sorting operations. During the last two rounds of selection, peptides with high affinity were selected by modifying the temperature of the wash solution (ambient temperature at 4 ° C) as well as the competitive temperature (ambient temperature at 4 ° C) and time (15-30 minutes). Using this affinity selection procedure, contiguous sequence YXCRIGPITWVC was screened for immobilized EPO-R (see FIG. 3). The fusion protein and the accompanying DNA were isolated and cultured to generate a fusion protein preparation and used for ELISA analysis to determine whether the fusion protein specifically bound to the receptor. Preferred peptides shown in this study are shown in Table 6.
GGEYICVMGPNTWVCSPTRGHGS GGEYLCRMGPMTWVSPFTRKGG GGQYICRFGPITWQSQPAGGGS GRAYSCRMGPITWVCMPRASLGS GDLYLCSMGITWICVPERGGGS ELWYSCRMGPVTWMCGRYQGGGS
In other variant studies, variant libraries with strongly conserved residues (ie, Y, C, G, P, T, and W) remained unmodified and other residues were randomized. Ten additional residues at the N-terminus preceding tyrosine were screened for immobilized EPO-R (see Figure 4A). Mutant fusion protein and the accompanying DNA were isolated and cultured to generate a fusion protein preparation and used for ELISA analysis to determine whether the fusion protein specifically bound to the receptor. Preferred peptides from this study are shown in Table 7.
QICRADRKGIYQCWYCPETWICgg QQGYSLWLPWYNCVLGPYTWVCgg YGGSAAVPWKYGCSLGPVTWVCgg QIVSWGLYSGYLCMVGPVTWVCgg GSGALSAAGWYGCRVGPLTWVCgg SVVSHDAAGVYDCVIGPVTWICgg IYSWTGILGSYVCWYGPDTWVCgg
SCIYVRFFYCYQCSEGPATWLCgg TVAKGQSGVRYSCLRGPETWVCgg VQPQYKWATMYQCWKGPSTWFCgg KSGVWEMGSSYQCARGPPTWCCgg CSVRRMDREYYRCCRGPFTWQCgg KYQEEMFMGYQCLQGPKTQLCgg VCPGSEFRVGYICAMGPYTWDCgg SLCSSRCNSPYFCSIGPSTWRCgg QASLGLPLKQYLCVLGPHTWLCgg ACKPAALFVQYGCVLGPMTWICgg SCERAGGRWEYVCQWGPDTWLCgg RVARQVQQVSYWCAHGPATCYCgg HKYDTLMLTNYVCQRGPLTQLCgg
In other variant studies, variant libraries with strongly conserved residues (ie, Y, C, G, P, T, and W) remained unmodified and other residues were randomized. Ten additional residues between the second cysteine and (GLY) -4-Ser linker were screened for immobilized EPO-R (see FIG. 4A). The tyrosine residues conserved in this library were located before two glycine residues to perform efficient signal peptide digestion. Mutant fusion protein and the accompanying DNA were isolated and cultured to generate a fusion protein preparation and used for ELISA analysis to determine whether the fusion protein specifically bound to the receptor. Preferred peptides from this study are shown in Table 8.
ggYHCEWGPETWICRPEISPLTVMgg ggYICDYGPLTWACKPAGATLLQPgg ggYTCRFGPVTWDCLPAINHNGVLgg ggYQ * FMGPETWVCAPEPRVERVSgg ggYLCRFGPETWTCAPERSVVTQSgg ggYVCDFGPTTWICRGQVMEHINTgg ggYMCNMGPLTWDCSPVRSTSMAWgg ggYHCEWGPETWICRPEISPLTVMgg ggYNCTMGPNTWVCTPAAESPAVFgg ggYGCRIGPITWICDDVSRSPRA ggYTCRMGPQTWECLPMSEGV ggYNCKFGPQTWDCSSANLKEV gYLCEMGPETWMCRPEDAKLGV ggYGCKFGPVTWICEDLLLDPMY ggYNCKFGPQTWDCSSANLKEVLV gYLCEMGPETWMCRPEDCEAW ggYGCGLAPVTWECPQVSIPYGLSgg ggYGCRIGPTTWICDSTVPQLREVgg ggYRCSWAPETWVCDNHSA gYLCNFGPITWDCVSSAQSEMQIgg
In another variant study, the library with strongly conserved residues (ie, Y, C, G, P, T and W) was unmodified, with tyrosine bound next to the first cysteine and 4 N for tyrosine. Other amino acids, including terminal residues, were randomized and screened for EPO-R immobilized 5 between the second cysteine and the linker (see FIG. 4C). The N-terminal random amino acid is again located ahead of the two glycine residues, allowing for an efficient process. The fusion protein and DNA encoding it were selected on immobilized EPO-R. The fusion protein and the accompanying DNA were isolated and cultured to generate a fusion protein preparation and used for ELISA analysis to determine whether the fusion protein specifically bound to the receptor. Preferred peptides from this study are shown in Table 9.
ggAELQYCKIGPETWVCDWPHIgg ggPYEGYCSGGPVTWECCVSVCgg ggQPLPYCSPGPTTWFCINWLFgg ggCSYGYCPMGPFTWMCRQRRLgg ggVRGSYCQSGPPTWQCDLRFFgg ggRCARYCACGPGTWNCLGRCQgg ggLGRCYCVYGPLTWWCSQTSLgg ggLCVWYCSAGPWTWYCIYRSAgg ggKPGPYCSFGPETWVCTALGMgg ggRLGEYCEIGPITWICRLFLPgg ggPGLGYCDFGPLTWVCDGSVDgg ggLSSAYCRYGPETWICWAGTGgg ggVLHLYCYYGPETWDCLPIKAgg ggGGGVYCLVGPVTWLCGPAAMgg ggLTRNYCRIGPETWICQEVAIgg ggWSERYCVLGPLTWECVHLFAgg ggMPLKYCGMGPVTWVCCEAVSgg ggSVMRYCHFGPETWICPYDMPgg ggALYPYCLICPMTWVCQVGWIgg ggTYGNYCRGGPGTWHCEDTRGgg ggASYCYCSKGPATWKCVGSILgg ggSLAAYCLQGPKTWPCVRRRLgg ggTDSLYCKLGPLTWHCQLYQKgg gISQQYCWRGPATWVCLEWELgg
In addition to the mutation studies described above, mutations of the peptide GGTYSCHFGPLTWVCKPQGG were performed using a modified C-terminal Lac-I display system with reduced display valency for the purpose of identifying high affinity hits (headpiece dimers). This Lac-I headpiece dimer (HPD) display system is described in detail in US Pat. No. 5,338,665. Indeed, the highly conserved residues (ie, Y, C, G, P, T, and W) in the aforementioned variation studies are slightly mutated while less conserved residues (ie, H, F, L, and V) Indicates a high rate of variation. Four random residues are located before the tyrosine residues and one random residue after the tyrosine. Behind cysteine is the variant C-terminus ending with 6 random amino acids. Detailed description of the resulting DNA construct is shown in FIG. 5.
The library was screened four times on EPO-R with PIG tail immobilized on mAb179, using the second EPO eluate to enrich the high affinity clones. The resulting DNA inserts were cloned and the pools were placed in maltose binding protein (MBP) vectors to allow them to be expressed as C-terminal fusion proteins. Crude lysates that were not purified from randomly selected individual MBP fusion clones were analyzed for EPO-R binding in ELISA format. Preferred peptides in this study are shown in Table 10.
RTKEYSCQMGPLTWICVPKS SKARYMCHMGPLTWVCRPEV GGKAYMCRLGPVTWVCSPRIKL LLRGYECYMGPLTWVCRSSKPR TIAQYICYMGPETWECRPSPDA NGRTYSCQLGPVTWVCSRGVRR LGRKYSCHFGPLTWVCQPAKKD MKTKYKCYMGPLTWVCEGS SKTKYRCEMGPLTWVCERW LTRLYSCHMGPSTWVCSTALRK RGQLYACHFGPVTWVCKRRKRV SGILYECHMGPLTWVCTPSRRR GSKTYSCQLGPVTWVCGRKR ARGKYQCQFGPLTWECLPIRPR VTRMYRCRMGPLTWVCER KPSLYECHLGPLTWECRRRRRE RGHMYSCQLGPVTWVCKPLSGR ITPTYHCKFGPQTWVCAPKRSALTK GNRMYQCHMGPLTWVCQPTRIH MKTKYKCYMGPLTWVCEGSPLK HLGKYDCSFGPQTWVCKRRRSL ERRVYECQMGPLTWECKPGVKG ITPTYHCKFGPQTWVCAPKRSALTK LGRKYSCHFGPVTWVCQPAKKD RGRGYSCQMGPVTWVCKRERYF RLREYRCHMGPQTWVCNGHHSK SGALYDCQMGPITWVCRANRQK TNQVYGCKFGPKTWVCKPARRI TRGMYACHMGPQTWVCRPTQPR VLSNYECTMGPKTWVCKPLRLK
One surprising feature of peptides obtained using the Lac-I headpiece langier display system is the accumulation of multiple positive charges in regions adjacent to conserved cysteines, especially RGQLYACHFGPVTWVCKRRKRV (which has a C-terminal stretch of five such residues). It is. Slightly mutated amino acids (ie, Y, C, G, P, T, and W) were fully preserved while new motive SMS were generated at the site of mutation. Of particular interest is the presence of additional tyrosine residues in the peptide number (LLRGYECYMGPLTWVCRSSKPR) and the presence of two glutamic acid residues present between cysteines in TIAQYICYMGPETWECRPSPKA.
In addition to the mutation studies described above, mutations of the peptide TISQYICYMGPETWECRPSPKA were performed using a C-terminal Lac-I display system similar to that described above. Indeed, the highly conserved residues (ie, Y, C, G, P, T, and W) in the aforementioned variation studies are slightly mutated while less conserved residues (ie, H, F, L, and V) Indicates a high degree of variation. The two glutamic acid residues were also slightly mutated. Four random residues are located before the tyrosine residues and one random residue after the tyrosine. Behind cysteine is the variant C-terminus ending with 6 random amino acids. A detailed description of the resulting DNA construct is shown in FIG. 6.
The library was screened three times on PIG labeled labeled EPO-R immobilized on mAb179, using the second EPO eluate to concentrate clones with high affinity. Collected colonies were used to demonstrate the human Fcγ-EBP reagent and the goat's antihuman Fcγ was conjugated to alkaline phosphatase, which was purchased from Sigma Chemical Company of St. Louis, Missouri, USA. Preferred peptides in this study are shown in Table 11.
ALKKYDCYFGPETWECLARRPH ERRFYKCRFGPETWECTL FGQEYRCHLGPETWQCSPVRVG FRPEYMCRMGPETWECGGARP GSRKYwCRMGPETWECMKPVRL GLKAYGCRYGPETWDCRSVILI IRQPYICHMGPETWECGRYPAG KGASYHCIMGPETWECIPQRVW MKQLYSCIMGPETWECRPGVER QRHYYRCALGPEYWECRPMSPE TKRLYHCHMGPETWECHGPMRK TRPSYRCAFGPVTWECIPAR PHKSYvCTFGPETWECTGAIRR RGRMYNCRMGPETWECKGQSKD RRRYYRCWMGPETWECSPVSNK VADNYDCPIGPVTWECIHVRAS VQKKYLCHFGPETWECGPDRD WQTWYICERGPETWECRWLVL YRMPYRCKMGPETWEVGGRGR YSREYSCRMGPETWECXRGFLR
The colonies of peptide sequences described above were cloned and placed in maltose binding protein (MBP) vectors to allow them to be expressed as C-terminal fusion proteins. Crude cell lysates, not purified from randomly selected individual MBP fusion clones, were assayed for EPO-R binding in ELISA format. Preferred peptides in this study are shown in Table 12.
RSMWYRCQMGPQTWVCGPRSAS SRREYICHLGPQTWVCGPGGRK GSPSYHCHLGPLTWVCKPHRMR MVGRQCHMGPRTWVCKPWHG GTARYQCHFGPLTWVCKPSLKG ELRGYICHFGPVTWVCKPNGSR LKQGYQCQLGPQTWVCRPLPMP KEPKYECQFGPRTWVCQPTRAN VRKVYACHMGPVTWVCVQGYKG SGRYVCRMGPETWVCRSYRGL ERRSYSCQMGPVTWVCGRQMGQ VKNNYRCQFGPVTWVCKAFR SGASYDCQMGPITWVCRANRQK
Representative peptides found to bind specifically to EPO-R were tested by cell lineage analysis. One of these assays used FDCP-1 as a parent cell line, which is a growth factor that depends on the rat multipotent primitive hematopoietic precursor cell line (eg Dexter et al., 1980, J. Exp. Med. 152: 1036-1047). Reference). This cell line is not diversified but can proliferate when supplemented with WEHI3-conditions medium (IL-3, medium containing ATCC T1B68). This was transfected with human or murine EPO-R as described below to generate FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively. These transfected cell lines proliferated but did not diversify in the presence of human or rat EPO.
Cells were incubated at a density of half the stationary density in the presence of the necessary growth factors. Cells were then washed in PBS and fed nothing to the whole medium without growth factor for 16-24 hours. After determining the viability of the cells, a stock solution with about 10 ⁵ cells (in total medium without growth factor) per 50 μl was made. Sequential dilutions of the compounds to be tested (typically solution phase peptides opposite to peptides that are phage bound or otherwise bound or immobilized) were made into 96-well tissue plates with a final volume of 50 μl per well. Cells (50 μl) are added to each well and cells incubated for 24 to 48 hours at which point the negative control should be killed. Cell proliferation is then measured by conventionally known techniques such as MTT assays involving incorporation of ³ H-thymidine as a measure of cell proliferation (Mosmann, 1982, J. Immunol. Methods 65:55). Figure 7 shows the analysis results for the peptides and EPO described in Table 5 above. Peptides that bind and exhibit activity in the ED receptor in FDCP-1 / hEPO-R cell line bioassays are preferred compounds of the invention.
The second cell line analysis used a TF-1 cell line (Kitamura et al., 1989, Blood, 73: 375-380). Representative results are shown in FIG. 8. 8 shows the effect of EPO and free peptide VGNYMCHFGPITWVCRPGGG (SEQ ID NO) on cell proliferation of cell line TF-1.
A third cell line assay showing the ability of the compounds of the present invention to function as EPO agonists is the incorporation of ³ H-thymidine into splenocytes of mice treated with phenylhydrazine. The results of this analysis are shown in FIGS. 9A-9C.
Other biological assays used to demonstrate the activity of the compounds of the present invention are disclosed in the following documents. Greenberger et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2931-2935 (EPO-dependent hematopoietic progenitor cell line); Quelle and Wojchowski (1991) J. Biol. Chem. 266: 609-614 (protein tyrosine phosphorylation in B6SUt.EP cells); Dusanter-Fourt et al. (1992) J. Biol. Chem. 289: 10670-10678 (tyrosine phosphorylation of EPO-R in human EPO-reactive cells); Quelle et al. (1992) J. Biol. Chem. 267: 17055-17060 (phosphorylation of cytostromal protein (pp100) in FDC ER cells); Worthington et al. (1987) Exp. Hematol. 15: 85-92 (color analysis of hemoglobin); Kaiho and Miuno (1985) Anal. Biochem. 149: 117-120 (hemoglobin detection using 2, 7-diamino fluorene); Patel et al. (1992) J. Biol. Chem. 267: 21300-21302 (transduction of c-myb; Witthuhn et el. (1993) Cell 74: 227-236 (binding and tyrosine phosphorylation of JAK2); Leonard et al. (1993) Blood 82: 1071-1079 ( the expression of GATA transcription factors); Ando etal (1993) Proc Natl Acad Sci USA 90:..... 9571-9575 (D2 and D3DM G 1/3 transition by cycling control); and calcium flux.
Using a device designed by Molecular Device Corp., known as a microphysiometer, it was possible to successfully measure agonists and antagonists for various receptors. This device measures changes in the rate of acidification of extracellular medium in response to receptor activation.
According to a preferred embodiment, the affinity and / or activity of the lead compound is increased by dimerizing or oligomerizing the peptides of the present invention. To determine whether the dimerization / oligomerization of peptides had EPO mimetic efficacy in cell proliferation assay, GGTYSCHFGPLTWVCKPQGG (Ahx-Biotin), a C-terminal biotinylated analog of GGTYSCHFGPLTWVCKPQGG, was synthesized.
This peptide was incubated with streptadidine in PBS at a molar ratio of 4: 1 for 1 hour at room temperature. Thereafter, PIG-labeled EPO-R binding assays were introduced to measure IC ₅₀ . The same experiment was conducted for peptides that did not bind streptavidin. The prebound peptide was found to have an IC ₅₀ of ₂₀ nM as compared to an IC ₅₀ 350 nM of peptides in which streptavidin was not incubated. The increased affinity is more than 10-fold presumably due to the polyvalent state of the peptide-streptavidin complex. Since this comparison was made using free peptide concentrations rather than effective complex concentrations (less than four times theoretically), the effect would be greater.
In addition, peptides were preincubated with streptavidin in serum-free HEPES-buffer RPMI at a molar ratio of 4: 1 as above. This complex was tested to stimulate cell proliferation in the FDCP-1 / hEPO-R Biological Assay Arongside Free GGTYSCHFGPLTWVCKPQGGSSK (Ahx-Biotin) and non-biotinylated peptides, ie GGTYSCHFGPLTWVCKPQGG. 10 is EPO-ED glass peptide ₅₀ but another similar (approximately 1μM), shows an analysis result showing a two log reduction in a less than that 10nM of streptavidin complex previously formed, i.e., EPO-ED _50. Streptavidin alone has only a slight irritant effect at the highest concentrations due to bacterial endotoxin contamination. In addition, there was an increase in biological potency when the peptide was dimerized with goat anti-biotin IgG. FIG. 11 shows that one log reduction in EPO-ED ₅₀ may be manifested by prior incubation of GGTYSCHFGPLTWVCKPQGGSSK (Ahx-biotin) and purified goat anti-biotin IgG at a molar ratio of 2: 1. This increase in biological potency is presumably due to the dimerization reaction of peptides by antibodies. Anti-biotin antibody alone had no effect on the cells. In addition, a 100-fold reduction in EPO-ED50 was seen in the GGTYSCHFGPLTWVCKPQGGSSK (Ahx-Biotin) -streptavidin complex. As such, by dimerizing or oligomerizing the lead peptides of the present invention, the affinity and / or activity of such peptides can be increased.
As another embodiment, the dimeric peptide analog of GGTYSCHFGPLTWVCKPQGG containing two disulfide bonds was prepared using the general scheme of FIG. 12. The first peptide chain was assembled on Tentagel resin. Fmoc-Lys (Alloc) was bound to the Knorr linker and the Alloc group was used as the protecting group located at right angles. Cys (Acm) was used as the first peptide chain. After completion of the first peptide chain, the Alloc group was removed and the second peptide chain was placed in the side chain amine of the lysine residue. Cys (trt) was used in this peptide chain. After the synthesis was completed, peptides were digested and purified from the resin. The peptide was then cyclized to a compound containing a single disulfide bond. The second disulfide bond was formed by iodine oxidation to yield a bicyclic dimer.
13 and 14 show EPO-R binding and biological activity of dimers in vitro. The affinity of dimers was tested for PIG labeled EPO-R immobilized on mAb179 in stripwells. Equilibrium binding analysis showed a 200-fold increase in affinity of the parent peptide GGTYSCHFGPLTWVCKPQGG at 200 nM and a 5-fold increase in affinity for the GGTYSCHFGPLTWVCKPQGGSSK (Ah _x -Biotin) -streptavidin complex. In vitro biological activity was increased by approximately 20-fold from ₅₀ nM (mopeptide) to 20 nM of EC _{50 as} determined by FDCP-1 / hEPO-R cell proliferation. As such, by dimerizing or oligomerizing the lead peptides of the present invention, the affinity and / or activity of these peptides could be significantly increased.
The peptides of the invention or derivatives thereof can be conjugated to a compound that binds to EPO-R to produce a compound that has more affinity for the receptor than when the conjugate consists of one of the compounds of the invention. The discovery of such peptides also facilitates the identification of peptides that bind to the same site on EPO-R, since the library or selection process can be selected to remove peptides with complementary non-blocking motifs.
Preferred motif sequences are provided as a means for determining the minimum size of an EPO agonist of the invention. The use of an encrypted synthetic library [ESL] is described in US patent application 946,239, filed September 16, 1992. This application is CIP application of 762,522, filed September 18, 1991. Or U.S. Patent Nos. 492,462, 624,162, filed December 7, 1990, and 805, 727, filed March 7, 1990, filed on a very large scale. Polymer synthesis systems are described in which one skilled in the art can only determine the minimum size of a peptide having such activity, but also form groups of peptides different from the desired motif (or the minimum size of the motif) at one, two or more residues. Any peptide can be made. Collected peptides were harvested to screen the ability to bind EPO-receptors. Such immobilized polymer synthesis systems or other peptide synthesis methods can be used to synthesize all cleavage analogs, all cleavage analogs, all cleavage and cleavage combinations of the compounds of the invention.
Peptides of the invention can also be prepared by classical methods known in the art, for example by standard solid phase techniques. Such methods include whole solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, and recombinant DNA techniques (see Merrifield, 1963, J. Am. Chem. Soc. 85: 2149). When used in solid phase, the synthesis starts from the C-terminus of the peptide using alpha amino protected resin. Suitable starting materials are prepared, for example, by attaching the required alpha-amino acids to chromomethylated resins, hydroxymethyl resins or benzhydrylamine resins. This chromomethylated resin is a trade name BIO-BEAD SX-1, available from Bio Rad Lavoratories, Richmond, California. Hydroxymethyl resin formulations are described in Bodonszky et al., 1966, Chem. Ind (London) 38: 1597. Benzhydrylamine (BHA) resins are described in Marshall, 1970, Chem.Commn.650, which were readily available in the form of hydrochloride from Beckman Instruments, Inc., Palo Alto, USA.
Therefore, the compounds of the present invention are prepared according to the method described in Gisin, 1973, Helv. Chim. Acta 56: 1467, for example by coupling an alpha-amino protected amino acid to a fluoromethylated resin in the presence of a cesium bicarbonate catalyst. Can be After initial coupling, the alpha amino acid protecting group is removed by reagents including trifluoroacetic acid (TFA) or hydrochloric acid (HCL) solution in organic solvent at room temperature.
Alpha-amino protecting groups are those known in the art for synthesis which proceed to the stage of the peptide. Examples thereof include acyl protecting groups (e.g. formyl, trifluoroacetyl, acetyl), aromatic urethane protecting groups (e.g. benzyloxycarbyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups (e.g. t -Butyloxycarbonyl, Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting groups (eg benzyl, triphenylmethyl). Fmoc is a preferred protecting group. Such side chain protecting groups (generally ethers, esters, trityl, PMC, etc.) remain undamaged upon coupling or do not cleave upon coupling or upon deprotection of amino-terminal protecting groups. These side chain protecting groups are removable when the synthesis of the final peptide is complete under conditions that do not modify the desired peptide.
The side chain protecting groups of tyrosine include tetrahydropyranyl, t-butyl, trityl, benzyl, methyl, ethyl and cyclohexyl. Side chain protecting groups of Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, Cbz, Z-Br-Cbz and 2,5-dichlorobenzyl. For Asp side chain protecting groups include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl. Side chain protecting groups of Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz. The side chain protecting groups of Thr and Ser are benzis. In the case of Arg, the side chain protecting groups include nitro, tosyl, Cbz, adamantyloxycarbonyl mestolsulfonyl (Mts), or Boc. For Lys the side chain protecting groups include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.
After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled step by step in the preferred order. Each protected amino acid is selected from 2- (1H-benzotriazol-1-yl) -1,1,3,3 tetramethyl in a solution (eg methylene chloride (CH ₂ Cl ₂ ), dimethyl formamide (DMF) mixture). Uronium hexafluorophosphate (HBTU) or dicyclohexylcarbodiimide (DCC).
After the desired amino acid sequence is completed, the preferred peptide is decoupled from the resin support by reagent treatment such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which not only degrades the peptide from the resin but also remains Decomposition of all side chain protecting groups. When chloromethylated resins are used, the hydrofluorination treatment forms free peptide acids. When benzhydrylamine resins are used, hydrogen fluoride treatment directly yields free peptide amides. As an alternative, when chloromethylated resins are used, the side chain protected peptides can be decoupled with ammonia by peptide treatment to produce side chain protected alkylamides or dialkylamides. Subsequent removal of the chain protection reaction according to the usual manner of treatment with hydrogen fluoride yields free amide, alkylamide or dialkylamide.
To prepare the esters of the invention, the resin used to make the peptide acid is used and the protected peptide is digested with a suitable alcohol such as base and methanol. Preferred esters are then obtained via conventional means in which the side chain groups are treated with hydrogen fluoride. These solid phase peptide synthesis procedures are known in the art and described in Stewart, Solid Phase Peptide Syntheses, Freeman and Co., Sanfrancisco, 1969.
The procedure can be used to prepare peptides in which 20 natural amino acids are substituted at one, two or more positions of any compound of the invention, other than those genetically encoded. In one example, naphthylalanine may be used in place of tryptophan for ease of synthesis. Other synthetic amino acids that may be substituted with the peptides of the invention include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, δ amino acids (e.g., L-δ-hydroxylsilyl and D-δ- Methylalanyl), L-α-methylalanyl, βamino acid and isoquinolyl. D-amino acids and non-natural synthetic amino acids can also be included in the peptides of the present invention.
Those skilled in the art will recognize that the naturally occurring side chains of the 20 genetically engineered amino acids (or D amino acids) are alkyl, lower alkyl, cyclic 4-, 5-, 6-, 7-membered alkyl, amide, amide lower alkyl, amide di Other side chains such as (lower alkyl), lower alkoxy, hydroxy, carboxy and lower ester derivatives thereof and 4-, 5-, 6-, 7-membered heterocyclic side chains. In particular, proline analogs in which the ring size of the proline residue is modified from 5-membered to 4-, 6- or 7-membered can be used. The cyclic group may be saturated or unsaturated and, if unsaturated, may be aromatic or non-aromatic.
Heterocyclic groups preferably include one or more nitrogen, oxygen and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl ( E.g. 1-piperazinyl), piperidinyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, Pyrimidyl, pyrrolidinyl (e.g. 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino) and thiazolyl Include. These heterocyclic groups may be substituted or unsubstituted. When the group is substituted, the substituent is alkyl, alkoxy, halogen, oxygen or substituted phenyl or unsubstituted phenyl.
One skilled in the art can readily modify the peptides of the present invention by phosphorylation, and other methods of preparing peptide derivatives of the compounds of the present invention are described in Hruby et al. Thus, the peptide compounds of the present invention are used to prepare peptide mimetics with similar biological activity.
Compounds of the invention, including peptide mimetics, can be covalently modified with one or more of various nonproteinaceous polymers such as polyethylene glycol, polypropylene glycol or polyalkenes. See for example, US Pat. No. 4,640,835; 4,496,698; 4,301,144; 4,670,417; 4,791,192; Or 4,179,337.
One of ordinary skill in the art appreciates that it is possible to use useful techniques for preparing peptide mimetics that have the same and similar preferred biological activities as the corresponding peptide compounds, but which have the preferred activity over peptides in solubility, stability and hydrolysis, and proteolysis. (See Morgan Gainor Ann. Rep. Med. Chem. 24: 243-252 (1988)). The following describes a method for preparing peptide mimetics that are modified at N-terminal amino groups, C-terminal carboxy groups and / or at least one amido bond in a peptide is modified to non-amido bonds. Two or more such modifications (eg, modification at the C-terminal carboxy group and incorporation of -CH-carbamate between two amino acids in the peptide) can be bound to one peptide mimetic structure.
Peptides are generally synthesized with free acid, but can be readily prepared as amides or esters as described above. One skilled in the art can also modify the amino and / or carboxy terminus of the peptide compounds of the invention to produce other compounds of the invention. Amino terminus modification methylation (i.e., -NHCH ₃ or NH (CH ₃ ) ₂ ), acetylation, addition of carbobenzoyl groups, or amino terminal with any blocker containing a carboxylate functional group that is RCOO- Reactions, wherein R is selected from the group consisting of naphthyl, acridinyl, stereoidyl, and similar groups. Carboxy terminus modifications include those having structural limitations by replacing the free acid with a carboxamide group or by forming cyclic ramtams at the carboxy terminus.
The amino terminal modification reactions are as described above and include alkylation reactions, acetylation reactions, carbobenzoyl group addition reactions, and succinimide group formation reactions. In particular, the N-terminal amino group can react as follows:
(a) reaction with an acid halide such as RC (O) Cl or an acid hydride to form an amide group of RC (O) NH—, where R is as defined above. Generally, the reaction is carried out by contacting an equimolar or excess (eg, about 5 equivalents) of the acid halide with the peptide in an inert diluent (eg, dichloromethane). This diluent is capable of scavenging the acid produced during the reaction and contains an excess of tertiary amine (eg diisopropylamine) (eg about 10 equivalents). Reaction conditions are normal conditions (for example, 30 minutes at room temperature). After lower alkyl N-substitution, the reaction with the above-described acid halides to alkylate the terminal amino provides an N-alkyl amide group of RC (O) NR-.
(b) A succinimide group is formed by reaction with succinic anhydride. As mentioned above, the use of equimolar or excess succinic anhydride (eg, about 5 equivalents) allows the conversion of amino groups to succinamides according to methods known in the art. The prior art refers to reacting an excess of tertiary amine (e.g., 10 equivalents) in an inert solvent (e.g. dichloromethane) in an inert solvent (see US Pat. No. 4,612,132 to Wolenberg et al.). The succinic groups can be substituted, for example, with C2-C6 alkyl or SR substituents substituted in a conventional manner to provide substituted succinimides at the N-terminus of the peptide. When the lower olefin (C2-C6) is reacted with maleic anhydride by the method of Wollenberg et al., Such an alkyl substituent is prepared, and the -SR substituent is RH with malee anhydride having the same meaning as described above. Manufactured by;
(c) animal or excess CBZ-Cl (e.g. benzyloxy carbonyl chloride) or substituted CBZ- in an inert solvent (e.g. dichloromethane) containing a tertiary amine that scavenges the acid produced during the reaction. Reacting Cl to form a benzyloxycarbonyl-NH group or a substituted benzyloxycarbonyl-NH group;
(d) equimolar or excess (e.g., 5 equivalents) of RS (O) ₂ Cl (R is as defined above) to convert the terminal amino group to sulfonamide by reaction in a suitable inert diluent (dichloromethane) To form sulfonamide groups; Preferably the inert diluent contains an excess of tertiary amine (10 equivalents), such as diisopropylethylamine, to scaveng the acid produced during the reaction. Reaction conditions are conventional (eg room temperature and 30 minutes).
(e) equimolar or excess (e.g., 5 equivalents) of R-OC (O) Cl or R-OC (O) OC ₆ H ₄ -p-NO ₂ (R is as defined above) in a suitable inert diluent ( Reaction in dichloromethane) converts the terminal amine groups to carbamate groups to form carbamate groups; Preferably the inert diluent contains an excess of tertiary amine (10 equivalents), such as diisopropylethylamine, to scaveng the acid produced during the reaction. Reaction conditions are conventional (eg room temperature and 30 minutes).
(f) by reacting an equimolar or excess (e.g., 5 equivalents) of RN = C = O (R is as defined above) with a suitable inert diluent (dichloromethane), the terminal amine group is urea (i.e., RNHC ( Is converted to an O) NH—) group (R is defined above) to form a urea group; Preferably the inert diluent contains an excess of tertiary amine (10 equivalents), such as diisopropylethylamine, to scaveng the acid produced during the reaction. Reaction conditions are conventional (eg room temperature and 30 minutes).
As an alternative, the C-terminus can be modified. In the case of preparing peptide mimetics when the C-terminal carboxyl group is replaced with an ester (-C (O) OR; R is defined above), the resin used to prepare the peptide acid is used and the side chain protected Peptides are degraded by bases and suitable alcohols (eg methanol). The side chain protecting groups are removed in the usual manner with treatment with hydrogen fluoride to obtain the preferred esters.
In preparing the peptide mimetic when the C-terminal carboxyl group is replaced with -C (O) NR ³ R ⁴ , the resin is used as a solid support for peptide synthesis. At the completion of the synthesis, hydrogen fluoride treatment to release the peptide from the support yields the free peptide amide directly (ie, when the C-terminus is C (O) NH) ₂ . Alternatively, the use of chloromethylated resins in the synthesis of coupled peptides by reaction with ammonia to degrade the side chain protected peptides from the support results in the formation of free peptides and the reaction of alkylamides with dialkylamides in the side chains. Protected alkyl amides or dialkylamides (R and R 1 are as defined above and the C-terminus yields —C (O) NRR ¹ ).
In another embodiment, the C-terminal carboxyl group or C-terminal ester may be derived to be cyclized by the internal arrangement of the ester having an N-terminal amino group or the carboxyl group -OH or ester (-OR) forming a cyclic peptide. . For example, after synthesis and degradation to peptidic acid, the free acid is ester activated by a carboxyl group such as dicyclocarbodiimide (DCC) in a solution (methylene chloride, CH ₂ Cl ₂ ), dimethyl formamide (DMF) mixture. Is switched to. The cyclic peptide is then formed by the internal configuration of the activated ester with the N-terminal amine. The internal cyclization reaction as opposed to polymerization can be improved by using very dilute solutions.
Those skilled in the art can incorporate desamino or desaccarboxy moieties at the ends of the peptides or cyclize the peptides of the present invention, thereby inhibiting the arrangement of the peptides without terminal amino groups or carboxy groups or reducing sensitivity to proteases. C-terminal functional groups of the compounds of the invention include amides, amide lower alkyls, amide di (lower alkyls), lower alkoxy, hydroxy, and carboxy, and lower ester derivatives thereof, pharmaceutically acceptable salts.
Another method of making peptide derivatives of the compounds of the present invention is described in Biochem J.268 (2): 249-262 (1990) by Hruby et al. Therefore, the peptide compounds of the present invention also serve as structural models of non-peptidic compounds with similar biological activity. Those skilled in the art will understand the techniques for preparing compounds having the same or similar biological activities as lead peptide compounds but having more activity than lead compounds in terms of solubility, stability and hydrolysis and sensitivity to proteolysis (Morgan Gainor , Ann. Rep. Med, Chem. 24: 243-252, 1989). These techniques include replacing the backbone of the peptide with a backbone consisting of phosphates, amide salts, carbonates, sulfonamides, secondary amines, and N-methylamino acids.
At least one peptidyl bond (-C (O) NH-) is a -CH ₂ -carbamate bond, a phosphonate bond, a -CH ₂ -sulfonamide bond, a urea bond, a secondary amine (-CH ₂ NH-) bond and Peptide mimetics replaced by bonds such as alkylated peptide bonds [-C (O) NR ⁶ , where R ⁶ is lower alkyl] are substituted by appropriately protecting amino acid analogs with amino acid reagents at appropriate points in conventional peptide synthesis. Are manufactured.
For example, suitable reagents include amino acid analogs in which the carboxyl groups of the amino acids are replaced with suitable moieties to form one or more bonds described above. For example, a -C (O) NR a peptide bond in the CH ₂ - carbamate bond _{(-CH 2 OC (O) NR-} ) -CH ₂ group in the first carboxyl (-COOH) of a suitably protected amino acid, if it substituted by Reduced to OH and then converted to -OC (O) Cl functional groups or para-nitrocarbonate -OC (O) OC ₆ H ₄ -p-NO ₂ functional groups according to conventional methods. The reaction of the functional group with either the free or alkylated amine on the N-terminus of the partially prepared peptide found on the solid support forms a CHO (O) NR bond. The reaction of such functional groups with free amines or alkylated amines on such CH ₂ -carbamate is described in Cho et al., Science, 261: 1303-1305, 1993, for details on the formation of bonds.
Similarly, substitution of phosphonate bonds with amido bonds of peptides can be performed according to the manner described in US Pat. Nos. 9943,805 and 119,700.
Amido bonds in the peptide can be replaced with -CH ₂ -sulfonamide bonds by reducing the carboxyl group (-COOH) of a suitably protected amino acid to -CH ₂ OH group and converting it to a suitable free group such as a tosyl group according to conventional methods. Can be. Reaction of the tosylated derivative with thioacetic acid followed by hydrolysis and oxidative chlorination yields a —CH ₂ —S (O) ₂ Cl functional group that replaces the carboxyl group of the amino acid that would have been adequately protected. A detailed description of the conversion of carboxyl groups of amino acids to -CH ₂ S (O) ₂ Cl groups is provided in Weinstein, Boris, Chemistry Biochemistry of Amino Acids, Peptides and Proteins, vol. 7, pp. 267-367, Marcel Dekker, Inc., New York (1983).
Amido bonds in peptides can be substituted with urea bonds in the manner described in US patent application Ser. No. 08 / 147,805.
Moderately protected dipeptide analogs in which carbonyl bonds of amido bonds are reduced to CH ₂ groups by conventional methods can be used to prepare secondary amine bonds in which the amido bonds in the peptide are replaced with -CH ₂ NH. For example, in the case of diglycine, reducing the amine to amide occurs after deprotection of H ₂ NCH ₂ CH ₂ NHCH ₂ COOH used in the N-protected form in the next coupling step. It is known in the art to prepare such analogs by reducing the carbonyl group of the amido bond at the dipeptide bond.
Properly protected amino acid analogs are used for conventional peptide synthesis in the same manner as used for the corresponding amino acids. For example, about 3 equivalents of the protected amino acid analog are used in this reaction. Inert organic diluents such as methylene chloride or DMF are used. When the acid is produced as a reaction byproduct, the reaction solvent generally contains an excess of tertiary amine to scaveng the acid produced during the reaction. Particularly preferred tertiary amines are diisopropylethylamines used in excess of about 10 times. The reaction results in incorporation into peptide mimetics of amino acids having non-peptidyl bonds. This substitution is preferably repeated so that everything from the absence of amido bonds in the peptide is replaced by non-amido bonds.
Those skilled in the art also cyclize the peptides of the present invention or mix desamino or decarboxy residues at the ends of the peptides to limit peptide arrangement without terminal amino groups or carboxyl groups or to reduce sensitivity to proteases. C-terminal functional groups of the compounds of the invention include amides, amide lower alkyls, amide di (lower alkyls), lower alkoxy, hydroxy, and carboxy, lower ester derivatives thereof and pharmaceutically acceptable salts thereof.
According to a preferred embodiment, residues X ₃ or X ₈ are each selected from C and Hoc groups. Thus, the compounds of the present invention have disulfide bonds in the molecule as cyclic. As an alternative, intramolecular disulfide bonds can produce dimer compounds. These intramolecular or intermolecular disulfide derivatives are represented by the following formula (1).
Wherein m and n are 1 or 2, respectively.
Another embodiment of the invention provides an analog of the disulfide derivative when one sulfur is replaced with a CH ₂ group or another isomer of sulfur. These analogues can be made from the compounds of the present invention, where X ₃ or X ₈ is C or Hoc and the other is alpha-amino-gamma-butyl acid, which are internal molecules or molecules according to the following known methods. We are mutually located.

In which p is 1 or 2. Those skilled in the art will fully appreciate that this arrangement can occur using other analogues of alpha-amino-gamma-butyl acid homocysteine.
In addition to the cyclization steps described above, other non-disulfide peptide cyclization steps can be used. Such alternative cyclization steps include, for example, amide-ringing steps, cyclization steps to form thio-ether bonds. Thus, the compounds of the present invention may exist in cyclized form by intramolecular amide bonds or intramolecular thio-ether bonds. To facilitate the amide-ring reaction step, a peptide, ggTYSCHFGPLTWVCKPQgg, is synthesized, wherein the first cysteine is replaced by lysine and the second cysteine is replaced by glutamic acid and cyclic through an amide bond between the two residue side chains. Monomers are formed. In addition, to facilitate the thio-ether cyclization step, a peptide, ggTYSCHFGPLTWVCKPQgg, is synthesized, wherein the first cysteine is replaced by lysine and a cyclic monomer via a thio-ether bond between these two residue side chains and the C-terminal cysteine. Is formed. As such, in addition to disulfide bonds, amide-ring reactions and thio-ether cyclization reactions can be readily used to cyclize the compounds of the present invention.
Alternatively, the amino-terminus of the peptide can be capped with alpha substituted acetic acid, where the alpha substituent is a free group, for example alpha-haloacetic acid (eg alpha-chloroacetic acid, alpha-bromoacetic acid or alphaiodoacetic acid). There is. Compounds of the invention wherein X ₃ is C or Hoc can be cyclized by disposing the free group with X ₃ residues as sulfur (Barker et al., 1992, J. Med. Chem. 35: 2040-2048 Or et al. 1991 J .Org.Chem. 56: 3146-3149).
The compounds of the present invention play a biological role in EPO, including assessing many of the factors affected and affected, binding EPO to EPO-R (e.g., EPO signal transduction / receptor activation), and producing EPO. It is useful as a unique in vitro tool for understanding. The present invention can also be usefully used in the development of other compounds that bind to EPO-R because the compounds of the present invention have an important structure-activity relationship (SAR) that facilitates such development.
Moreover, given their ability to bind EPO-R, the compounds of the present invention are useful as reagents for detecting EPO-R in living cells, immobilized cells, biological fluids, tissue homogenates, purified natural biological materials, and the like. . For example, by labeling these peptides, cells with EPO-R on their surface can be identified. In addition, based on their ability to bind EPO receptors, the peptides of the present invention are capable of in situ staining, namely FACS (Shape-Activated Cell Salting), Western Blotting, and ELISA (Enzyme-linked Immunosorbent Assay). . And based on their ability to bind to EPO receptors, the peptides of the present invention can be used to purify receptors, or to purify cells expressing EPO-R at the cell surface.
The compounds of the present invention can be used as commercial reagents that can be used for various medical research and diagnostic purposes. Such uses include, but are not limited to, the following: 1) Use as a standard to measure the amount of activity of potential EPO agonists in various functional assays; 2) use as a blocking reagent in random peptide screening (ie, in finding a new group of EPO-R peptide ligands, the peptide can be used to block the recovery of currently claimed EPO peptides); 3) use as co-crystallization with EPO-R (ie, peptides of the invention form crystals that bind to EPO-R to facilitate measurement of receptor / peptide structure X-ray crystallography); 4) measuring the ability of erythrocyte precursor cells to vary and stimulate globin synthesis and to result in the synthesis of ham complexes and an increase in the number of ferritin receptors; 5) use to maintain proliferation and growth of EPO dependent cell lines such as FDCP-1-mEPO-R TF-1 cell line; 6) Other research and diagnostic purposes for the purpose of conveniently activating EPO-R or for measuring this activity conveniently for known amounts of EPO agonists and the like.
The compounds of the present invention can be administered to calm animals, including humans, to stimulate the binding of EPO-R in vivo. Accordingly, the present invention encompasses a method for the treatment of a disease in which an amount of a compound of the present invention is sufficient to stimulate EPO-R to alleviate symptoms associated with binding of EPO in vivo. For example, the compounds of the present invention may be used to treat terminal renal failure / dialysis; Anemia associated with AIDS, anemia associated with chronic inflammatory diseases (eg, rheumatoid arthritis and chronic enteritis), autoimmune diseases, malignancies; And for injecting red blood cells into a patient prior to surgery.
According to another embodiment of the present invention, the compounds of the present invention are also used in the treatment of diseases that do not have low or deficient numbers of red blood cells as pretreatment for transfusion. In addition, the administration of a compound of the present invention reduces the bleeding time and thus provides indications for which bleeding will occur or for use in a patient prior to surgery. In addition, the compounds of the present invention have the use of activating megakaryocytes.
EPO has been shown to have mitotic and chemotactic effects on vascular endothelial cells and cholinergic neurons (eg Amagnostou et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5978-5982 and Konishi et al., 1993, Brain). Res. 609: 29-35), the compounds of the present invention treat various vascular diseases, for example, wound healing, growth of collateral vessels (which can occur after myocardial infarction), trauma, after vascular junction therapy. It can be used to treat a variety of neurological diseases (due to a relatively low amount of acetylcholine or an absolutely low amount of acetylcholine when compared to other neuroactive agents, neurotransmitters).
Accordingly, the present invention encompasses pharmaceutical compositions containing as active ingredient at least one peptide or other compound of the present invention in connection with a pharmaceutical carrier or diluent. Compounds of the invention can be used orally, parenterally (intravenously, intraperitoneally, intramuscularly, or subcutaneously), intradermal (passively or by ion osmotherapy, by electroporation), intramucosal root (nasal, vaginal, rectal). Or sublingually) or in the form of a biologically edible insert and formulated in a dosage form suitable for each route of administration.
Solid dosage forms suitable for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage formulations, the active compound is mixed with one or more uncertain pharmaceutically acceptable carriers such as sucrose, lactose, or starch. It also contains, in accordance with conventional practice, additional components other than reactants such as magnesium stearate, inert diluents (lubricants). In the case of capsules, tablets and pills, the dosage form contains a buffer, a reactant. Tablets and pills can be enteric coated.
Liquid formulations suitable for oral administration include inert diluents commonly used in the art of pharmaceutical emulsions, solvents, and syrups, for example, elixirs containing water. In addition to these diluents, the formulation contains auxiliaries such as wetting agents, emulsifiers, suspending agents, sweetening agents, spices or flavorings.
The preparations according to the invention contain sterile aqueous or non-aqueous solvents, suspensions, or emulsifiers. Such non-aqueous solvents or media include propylene glycol, polyethylene glycol, vegetable oils such as injectable organic esters such as olive oil, corn oil, gelatin and ethyl oleate. Such formulations contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. For example, they can be filtered through a bacterial retention filter, mixed with a sterile agent into the composition, irradiated with the composition, or prepared using other sterile injection media just prior to use.
Compositions suitable for rectal administration and vaginal administration are preferred suppositories containing the active ingredient together with excipients such as cocoa butter or suppository waxes. Nasal or sublingual administration is prepared by standard excipients known in the art.
The amount of active ingredient to be added to the composition of the present invention may vary, but the amount of active ingredient is that amount in which a suitable dosage formulation is obtained. The choice of dosage depends on the desired therapeutic effect, route of administration and duration of treatment. It is generally administered to mammals in an amount of 0.001-10 mg body weight per day.
As disclosed above, the present invention has several uses. The examples are described below for non-limiting embodiment purposes.
As a first aspect, the present invention provides a peptide that binds to and activates EPO-R or otherwise acts as an EPO agonist. These peptides consist of amino acid residues 10 to 40 or more in length, preferably 14-20 amino acid residues in length, which represent the amino acid core sequence X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ (SEQ ID NO: Wherein each amino acid is a standard term abbreviation and has the following meaning: X ₃ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc (Hoc is homocysteine); X ₄ may be R, H, L, or W; X ₅ is M, F, or I; X ₆ is each selected from any of twenty genetically encoded L-amino acids or stereoisomer D-amino acids; X ₇ is D, E, I, L, or V; X ₈ is C, or Hoc (Hoc is homocysteine) provided that X ₃ or X ₈ is either C or Hoc. The peptide preferably comprises the YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ (SEQ ID NO :) Ko sequence, where each amino acid has the following standard abbreviations: X ₂ and X ₆ Are each selected from any one of 20 genetically encoded L-amino acids; X ₃ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc (Hoc is homocysteine); X ₄ may be R, H, L, or W; X ₅ is M, F, or I; X ₇ is D, E, I, L, or V; X ₈ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc (Hoc is homocysteine) provided that X ₃ or X ₈ is either C or Hoc.
More preferably the peptide comprises the core sequence X ₁ YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ X ₉ X ₁₀ X ₁₁ (SEQ ID NO), wherein each amino acid is indicated by standard abbreviation It has the following meaning. Each X _1, X _2, X _6, X _9, X _10, X ₁₁ is selected from any of 20 genetically encoded L-amino acids; X ₃ is C, A, alpha-amino-gamma-bromobutyl acid, or Hoc, where Hoc is homocysteine; X ₄ may be R, H, L, or W; X ₅ may be M, F, or I; X ₇ may be D, E, I, L or V; X ₈ may be C, A, alpha-amino-gamma-bromobutyl acid, or Hoc (where Hoc is homocysteine) provided that X ₃ or X ₈ is C or Hoc.
In a more preferred embodiment, since X ₃ and X ₈ are C, the peptide comprises the core sequence X ₁ YX ₂ CX ₄ X ₅ GPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO) of the amino acid. More preferably, the peptide comprises the core sequence X ₁ YX ₂ CX ₄ X ₅ GPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO) of the amino acid, wherein X ₄ or R or H; X ₅ is F or M; X ₆ is I, L, T, M or V; X ₇ is D or V; X ₉ is G, K, L, Q, R, S, or T and X ₁₀ is A, G, P, R or Y. In the most preferred embodiment, the peptide comprises the core sequence X ₁ YX ₂ CX ₄ X ₅ GPX ₆ TWX ₇ CX ₉ X ₁₀ X ₁₁ (SEQ ID NO) of the amino acid, wherein X ₁ is D, E, L, N, S , T or V; X ₂ is A, H, K, L, M, S or T; X ₄ is R or H; X ₉ is K, R, S or T; X ₁₀ is P.
Particularly preferred peptides include, but are not limited to:

In a preferred embodiment, the peptides of the invention are cyclized or dimerized. Examples of peptides that can be cyclized as C-terminal amides include the following. This is merely an example and the present invention is not limited to this:

Examples of dimers of the invention are as follows:

According to an embodiment of the invention, two or more, preferably 2-6 amino acid residues each selected from any one of twenty genetically-encoded L-amino acids or stereoisomer D-amino acids, one terminal or sock of the core sequence described above Coupled to the stage. For example, the sequence GG is attached at the end or at the end of the core sequence to facilitate the synthesis of the peptide. The present invention provides peptide peptidomimetic of peptides possessing the properties of EPO-R binding and conjugates of these peptides and derivatives thereof.
The present invention also provides methods for treating diseases including defects of EPO using novel compounds. The present invention further provides pharmaceutical compositions containing one or more compounds and a pharmacologically acceptable carrier.
Example 1
Solid Phase Peptide Synthesis
Merrifield solid phase synthesis techniques (Stewart, JM, and Young, JD, Solid Phase Pepide Synthesis, 2d. Edition, Pierce Chemical, Rockford, IL, 1984) on a Milligen / Biosearch 9600 automated machine were used to Synthesized. The resin used was PAL (Milligen / Biosearch), which is a polyester, crosslinked by 5- (4'-Fmoc-aminomethyl-3,5'-dimethoxyphenoxy) valeric acid as a linker. The use of PAL resins results in carboxy terminal amide function when peptides are degraded from the resin. The protection of the major amines on amino acids is achieved by F-moc, the side chain protecting groups are t-butyl for serine and thyroxine hydrolysis, trityl for glutamine amide, Pmc (2,2,5,7, 8-pentamethylchroman sulfonate) guanidino group. Each coupling was performed for 1 or 2 hours with BOP (benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate) and HOBot (1-hydroxybenztriazole).
In peptide synthesis with amidated carboxy terminus, a fully bound peptide was increased to 90% trifluoroacetic acid, 5% ethanedithiol at a temperature initially increased to 4 ° C. and then gradually to room temperature over 1.5 hours. And a mixture of 5% water. The deprotected product from the resin was filtered off and then precipitated with diethyl ether. After complete drying, the product was purified by C18 reverse phase high performance liquid chromatography with a gradient of acetonitrile / water in 0.1% trifluoroacetic acid.
Example 2
Biological analysis
A. FDCP-1 / mEPO-R
FDCP-1 / mEPO-R cells (10 ⁵ ) were incubated at a density of half the stationary phase in the presence of growth factor (2.5 nm EPO). The cells were washed twice with PBS and nothing was added to the whole medium for 24 hours without growth factors.
Cell viability was measured by trypan blue staining. The stock solution in the whole medium without growth factor was allowed to have a predetermined number of cells per 50 µl. Compounds to be tested were diluted 2-fold in 96 well tissue culture plates (50 μl per final well).
Cells (50 μl per well) were added to each well and incubated for 24-48 hours (when the negative control died). Cell proliferation was measured by MTT assay.
B. TF-1
The method described in Kitamura et al. (Blood 73: 375-380 (1989)) associated with cell line TF-1, which depends on EPO growth, was performed to demonstrate the activity of the compounds of the present invention as EPO agonists. The result is shown in FIG. 8 shows the effect of EPO and peptide VGNYMCHFGPITWVCRPGGG on cell proliferation of cell line TF-1.
C. Splenocyte Proliferation
Kristal (Exp. Hematol. 11: 649-660 (1983)) was used for microscopic analysis to assess the incorporation of ³ H-thymidine into splenocytes to confirm the ability of the compounds of the present invention to serve as EPO agonists. That is, B6C3F ₁ mice were administered daily with phenylhydrazine (60 mg / kg) for 2 days On the third day, splenocytes were isolated and their ability to proliferate over 24 hours using MTT assay was verified. Pictures taken at 200 magnification showing representative numbers of splenocytes proliferating are shown in FIG. 9A (control), 9B (treated with 500 pM EPO), and FIG. 9C (25 μL GGDYHCRMGPLTWVCKPLGG).
D. Cellular Proliferation: Peptide-dependent Growth of EPO-Reactive Cell Line, FDC-P1 / ER
1. Materials and Methods
a. Sample Preparation: Peptides were resuspended as 1 x 10 ^-2 M stock solution in DMSO and serially diluted in 10-fold increments to produce the following 100-fold solution:
1x10 ^-3 1x10 ^-4 1x10 ^-5 1x10 ^-6 1x10 ^-7 1x10 ^-8
2 μl of each stock dilution was added to the cell well as described below to give a final volume of 200 μl to produce the following concentrations:
1x10 ^-5 1x10 ^-6 1x10 ^-7 1x10 ^-8 1x10 ^-9 1x10 ^-10
b. Cell Proliferation Assay:
i. Cell source: FDC-P1 / ER (Dexter et al. J. Exp. Med. (1980) 152: 1036-1047) is characterized by a cell line derived from bone marrow of untransformed rats in which EPO-R was stably truncated. Well done. The cells exhibit EPO dependent proliferation.
c. Experimental Protocol: RPMI 1640/10% bovine fetal serum / antibiotic and cells maintained in 10 U / ml EPO were cultured in stationary phase. Cells were harvested and resuspended in fresh medium without growth factor (EPO) for 24 hours. Cell numbers were counted and resuspended at a concentration of 800,000 cells / ml. About 40,000 cells (50μ) were added to each well of a 96 well microtiter plate. Peptide was added to each well three times. Standard dose response measurements were taken for each series of peptides. After incubation for 42 hours (after twice the number), each well was pulsed with 1 μL Ci / well thymidine. Cells were incubated for 6 hours, at which point cells were harvested and counted and thymidine incorporation was assessed as evidence of cell proliferation.
d. Results: Peptides against EPO receptor cell lines and parent cell lines with non-receptors were evaluated. In most cases, peptides were evaluated against cleaved human EPO receptor cell lines. The results were expressed as the amount of peptide needed to obtain 1/2 the maximum activity obtained by recombinant EPO. Representative results are shown in the table as relative binding data (see Tables 17-22).
E. Tyrosine Phosphorylation: Tyrosine Phosphorylation Induced by EPO-R and Peptides of Intracellular Proteins
1. Materials and Methods
a. Sample Preparation: Peptides were suspended in DMSO to obtain a concentration of ¹ × 10 ⁻² M.
b. Experimental protocol: RPMI 1640/10% bovine fetal serum / antibiotic and FDC-P1 / muER cells maintained in 10 U / ml EPO were cultured in stationary phase. Cells were harvested and resuspended in fresh medium for 24 hours without growth factor (EPO). Cell counts were counted and resuspended at a concentration of 500,000 cells / ml. 1 ml of cells (500,000) were placed in an eppendorf tube. 1 μl of 1 × 10 ⁻³ peptide solution was added to the cells (final concentration 1 × 10 ⁻⁵ M) and incubated at 37 ° C. for 10 minutes. EPO controls (final concentration 10 U / ml) were analyzed respectively. Cells were harvested by centrifugation at 4 ° C. at 14,000 rpm. The cells were resuspended in 100 μl SDS lysate buffer and subjected to SDS polyacrylamide gel electrophoresis. The gels were transferred to nitrocellulose and probed with antiphosphotyrosine antibody (purchased from Upstate Biotechnology Incorporated) diluted 1: 1000 fold for 1 hour. Membranes were washed with Tris buffer saline and reprobed with anti-mouse peroxidase labeled antibody. Reactive protein was visualized using ECL western blotting reagent (Amersham).
c. Results: EPO binding to its receptor in EPO-reactive cell lines induces tyrosine phosphorylation on both receptors and many intracellular proteins including Shc, vav and JAK2 kinases. Analyzing a number of peptides, including GGTYSCHFGPLTWVCKPQGG, was able to assess their ability to mimic the response exhibited by EPO. As judged by binding criteria and proliferation criteria, the active peptide elicited the same phosphorylation pattern of EPO in EPO-reactive cells. These results suggest that the active peptides elicit their responses through the EPO-induced signal transduction pathway. The results are shown in Table 17-22 as binding data and propagation data.
F. Peptide-Induced Initiation of Specific Cell Cycles with DNA Components
1. Experimental protocol: FDCP-1 / muER cells were cultured to stationary phase at about 3.0 × 10 ⁶ cells / ml. Cells were harvested, suspended in fresh vegetation in the absence of growth factors and resuspended for 18 hours, at which point the cells were divided into three flasks with the same cell density: -factor, + EPO and + peptide. Cells were then stimulated with 10 U / ml EPO or 10 μM peptide. Three million cells were harvested at time points 0, 6, 8, 10 and 12 hours (FIG. 15). Cells were placed in cold PBS, washed twice, and cell pellets were resuspended in 100 μl cold PBS and resuspended with 900 μl cold 70% ethanol. After staining the cells with 50 μl / ml propidium iodine Fluorescence was measured by FACS Scan Flow cytometer. The content of cells in each phase (%) was measured by Becton Dickinson using the SOBR model CellFIT software.
EPO (10 U / ml) and GGTYSCHFGPLTWVCKPQGG (1 × 10 ⁻⁵ M) had the same effect on cells growing through the cell cycle. By induction for 10 hours with either EPO or peptide, about 45% of cells were placed on S compared to 15% media control. These results suggest that the peptide can induce a reaction with the same kinetic as recombinant EPO.
G. Colony Analysis: Analysis of Bone Marrow and Human Peripheral Blood Colonies
1. Materials and Methods:
A 10 ml sample of peripheral blood in a standard sterile heparin vacuum tube was obtained from a healthy individual. Rat bone marrow was obtained from the femurs of about 10 mice per experiment. All reagents were purchased from Stem Cell Technologies Inc (Vancouver, Canada).
a: experimental prototype; Nucleated cell number measurement is intended to determine the number and concentration of nucleated cells in the original sample. For such peripheral blood, centrifugation was performed on the samples using a 400 g Ficoll-Hypaque (1.077 g per ml) gradient at room temperature for 30 minutes. The mononuclear cells were carefully removed from the crab face of the Ficoll-Hypaque solution and diluted to a total volume of about 10 ml. Cells from rat bone marrow or human peripheral blood were harvested by centrifugation and supernatants were followed. The pelleted cells were resuspended in fresh medium and harvested as described above. Washed cells were diluted in serum of Iscove's medium / 2% bovine serum at the following plate concentrations.
Normal Bone Marrow: 1x10 ⁵ Low Density Cell Count
Normal blood: 4x10 ⁵ low density cell count
Cells were added to methyl cellulose according to the manufacturer's instructions. 1 × 10 ⁻⁶ and 1 × 10 ⁻⁵ M. peptides were analyzed in methyl cellulose medium without EPO. To assess the maximum colony formation, the same cell number was plated in complete methyl cellulose medium. Each assay without EPO and peptide was run on a control plate to determine if colonies were formed. Colonies were counted after incubation for 10 and 18 days.
b. Results: As shown in Table 13-15, GGTYSCHFGPLTWVCKPQGG could promote colony formation, despite the significantly lower concentrations of colonies compared to those obtained in complete methyl cellulose medium (EPO) in rat bone marrow and human peripheral blood. there was.
Colony Analysis on Human Peripheral Blood Specimens (94.10.2. Setup) datesampleCFU-EMBFU-EPBFU-ECFU-GM 2/23/94ZEROXXXXDMSOXXXXEPO (METHOCULT)1125XXEPO (3U / ml)77XXAFFY 244 10 ^-5 M105XXAFFY 244 10 ^-6 M115XX 3/4/94 ZEROXXXXDMSOXXXXEPO (METHOCULT)One11139EPO (3U / ml)4773AFFY 244 10 ^-5 M7924AFFY 244 10 ^-6 M611One2 CFU-E: Colony-forming unit-red blood cells (1-2 clusters) MBFU-E: Aged erythrocyte forming unit- red blood cells (1-3 clusters) CFU-GM: granulocyte / monocyte / macrophage of colony forming unit
Colony forming cell count (in the bone marrow of mice) 1/28/94 2/10/94PrecursorControlEPO10 ^-5 M10 ^-6 MCFU-E02323BFU-E0500CFU-GM03002/21/94PrecursorControlEPO10 ^-5 M10 ^-6 MCFU-E0000BFU-E03024CFU-GM0300 AFFY11157 = ggTYSCHFGPLTWVCKPQgg
Colony forming cell count (in the bone marrow of mice) 2/21/94 3/1/94PrecursorControlEPO10 ^-5 M10 ^-6 MCFU-E0390BFU-E03120CFU-GM01000 3/14/94PrecursorControlEPO10 ^-5 M10 ^-6 MCFU-E00One0BFU-E01080MBFU-GM0040CFU-GM02600
H. Peptide-Induced Proliferation: Peptide-dependent Growth on Cell Lines Not Responding to EPO
a. Experimental Protocol: Peptides were analyzed as described in section D above. To assess the growth potential, growth curves were plotted on each cell line using the relevant mitosis / growth factor for each cell line.
b. Results: As shown in Table 16, GGTYSCHFGPLTWVCKPQGG was unable to stimulate growth responses on non-hematopoietic cell lines, including hematopoietic and non-hematopoietic cell lines.
Cell Proliferation Assay Using 61233 Cell typeSpecimenEPO61233Growth factor response Hematopoietic cells TF-1human++GM-CSF, IL3 (RBC) M-07Ehuman--GM-CSF, IL3 (megakaryocytometer) AML193human--GM-CSF, G-CSF, IL3 (Monocytes) T-cell clonehumanND-IL2 Osteoblast TE85human--serum MC3T3rat--serum Chest cells T47DhumanND-Progestin
I. Competitive binding analysis of I ¹²⁵ ηEPO, the main component of immobilized EPO RK
The extracellular domain of the human EPO receptor (EPO binding protein, EPB) was expressed in E. coli and mass produced. this. As in the case of many other recombinant eukaryotic proteins used in Collie, the protein appeared to be an insoluble product in the laboratory scale fermentation process, which was then folded and purified to obtain the active protein. The EBP produced by this method contains one free sulfhydryl group that can be modified without effecting solution phase binding of the ligand. In order to immobilize the EPB as a basis for the equilibrium binding assay and the competitive binding assay, it was observed whether the EPB covalently bound to the agarose beads. It is clear that the iodoacetyl activity chemistry of Sulfolink beads (Pierce Chemical Co, Rockford, IL) is specific for free thiols and the binding is not readily reversible. EBP-Sulfolink beads were made as follows: Sulfolink gel suspension (10 ml) was mixed with coupling buffer (40 ml: 50 mM Tris, pH 8.3, 5 mM EDTA) and the gel precipitated. The supernatant was separated and then EBP (0.3 mg / ml in coupling buffer) to be bound was added directly to the washed beads. The mixture was gently shaken at room temperature for 30 minutes and the beads were allowed to stand at room temperature for 1 hour. The supernatant was separated and retained and the beads were washed twice with 20 ml of coupling buffer. The wash was also recovered. The beads were then treated with 20 mM 0.05 M cysteine at room temperature for 30 minutes to block unbound sites. Finally, the beads were washed with 50 ml of 1 M Nacl followed by 30 ml of PBS, resuspended in 20 ml of PBS and stored at 4 ° C. for later use. The amount of EBP covalently bound to the beads was determined by comparing the OD ₂₈₀ value of the original EBP solution with the OD ₂₈₀ value recovered from the reaction supernatant and two 20 ml washes. In general, 40-60% of the applied EBP remained sifted with the beads.
EBP beads (50 μl) were added to individual reaction tubes to initiate binding assays. Total binding was measured in tubes containing 0.3-30 nm [ ¹²⁵ I] -EPO (NEN Research Products, Boston MA, 100 μCi / μg). To measure nonspecific binding, non-labeled EPO was added in an amount 1000 times the corresponding [ ¹²⁵ I] EPO concentration. Binding buffer (PBS / 0.2% BSA) was used to bring each reaction volume to 500 μl. The tubes were incubated for 5 hours with gentle stirring at room temperature (this time determined experimentally as a suitable time to equilibrate). After 5 hours, each reaction mixture was passed through a 1 μl pipette tip sealed with glass wool. The tube was washed with 1 μl wash buffer (PBS / 5% BSA) and this and two 1 μl washes were passed together through a pipette tip and collected to determine the concentration of free EPO. Equilibrium binding assay for the specific binding of EBP immobilized on these agarose beads and [ ¹²⁵ I] EPO showed a Kd of 5 nm ± 2 based on linear transformation of binding isotherms (see FIG. 16). ).
Competitive binding assays of potential peptides were performed as follows. Each peptide was dissolved in DMSO to prepare 1 μM stock solution. All reaction tubes (2 each) contained 50 μl EBP beads, 0.5 nM ¹²⁵ I] EPO and 0-500 μM peptide in 500 μl binding buffer. The final concentration of DMSO was adjusted to 2.5% in all assay tubes, which showed no detectable effect. The reason for this was that the concentration of DMSO (V / V) of less than 25% had no detrimental effect on binding. Non-specific binding was measured by each population analysis using a tube containing an unlabeled EPO (1000 nM) excess. Initial assay points without added peptides were included to measure celestial binding in each assay. The binding mixture was incubated overnight at room temperature with gentle stirring at room temperature. Beads were then collected using Micro-Column (Isolab, Inc., Akron, Ohio) and washed with 3 mL wash buffer. The column containing the washed beads was placed in a 12 × 75 mm glass tube and the bound radioactivity level was measured using a gamma counter. The amount of bound [ ¹²⁵ I] EPO was expressed as% by adding control (total = 100%) binding to peptide concentration after correcting for non-specific binding. IC ₅₀ was defined as the binding of [ ¹²⁵ I] EPO to EBP. All data was recorded for GGTYSCHFGPLTWVCKPQGG (RWJ61233), which showed an IC ₅₀ of 5 μM (see Table 17-22).
GGTYSCHFGPLTWVCKPQGG substitution series SEQ ID NO:orderRelative Coupling ^‡ EPO-ED ₅₀ (μM)Phosphorylation ¹61233 ^‡ GGTYSCHFGPLTWVCKPQGGOne0.1+ 61231GGTASCHFGPLTWVCKPQGG24IA- 61520GGTTSCHFGPLTWVCKPQGG24IA- 61598GGTFSCHFGPLTWVCKPQGG12IA- 61277GGTYSCHFGALTWVCKPQGG20.1+ 61278GGTYSCHFGPLAWVCKPQGG18IA- 61213GGTYSCHFAPLTWVCKPQGG16IA- 61314GGTYSCHFGPATWVCKPQGGOne.2+ 61395GGTYSCHFGPLTAVCKPQGG100IA- 62145GGTYSCHFGPLTFVCKPQGG60.2561530GGTYSC-FGPLTWVCKPQGG40IA-^* Amount required to reach maximum level 1/2 (11 pM) of EPO dependent proliferation reaction ¹ Analyzed at 10 μM ² IA = inactive ^‡ Binding to RWJ-61233 ^‡ All peptides are cyclic (except 61394) and COOH termini Analyzed as amide (-CONH ₂ ) 61233 = AFFY1157 = AFFY244
GGTYSCHFGPLTWVCKPQGG Cutting Series SEQ ID NO:orderRelative Coupling ^‡ EPO-ED ₅₀ (μM)Phosphorylation ¹61233 ^‡ GGTYSCHFGPLTWVCKPQGGOne0.1+ 61596GGTYSCHFGPLTWVCKPQ1.60.08+ 61597TYSCHFGPLTWVCKPQGG82+ 61230TYSCHFGPLTWVCKPQ62+ 61232YSCHFGPLTWVCKP920+/- 61279YSCHFGPLTWVCK143+/- 61477YSCHFGPLTWVC50IA ² - 61895YSCHFGALTWVCK32IA61513Y-CHFGPLTWVC122+ 61177SCHFGPLTWVCK18IA- AF11154GGCRIGPITWVCGG13IA- 61394HFGPLTWV100IA-^* Amount required to reach maximum level 1/2 (11 pM) of EPO dependent proliferation reaction ¹ Analyzed at 10 μM ² IA = inactive ^‡ Binding to RWJ-61233 ^‡ All peptides are cyclic (except 61394) and COOH termini Analyzed as amide (-CONH ₂ ) 61233 = AFFY11157 = AFFY244
Analog sequence of peptide SEQ ID NO:orderRelative Coupling ^‡ EPO-ED ₅₀ (μM)Phosphorylation ¹61233 ^‡ GGTYSCHFGPLTWVCKPQGGOne0.1+ 61721 100IA ² 61718GGLYACHMGPMTWVCQPLRG0.60.161719GGEYLCRMGPMTWVCTPVGG8961720GGLYTCRMGPITWVCLPAGGND-61717GGNYYCRFGPITFECHPTGG100 (S1)IA ^* Amount required to reach maximum level 1/2 (11 pM) of EPO dependent proliferation reaction ¹ Analyzed at 10 μM ² IA = inactive ^‡ Binding to RWJ-61233 ^‡ All peptides are cyclic (except 61394) and COOH termini Analyzed as amide (-CONH ₂ ) 61233 = AFFY11157 = AFFY244
SEQ ID NO:(X)orderRelative Coupling ^‡ EPO-ED ₅₀ (μM) ^*Mouse receptorsCleaved human receptor 61233 ^‡ GGTYSCHFGPLTWVCKPQGGOne0.10.1 61231 GGTASCHFGPLTWVCKPQGG24IA ² 61520 GGTTSCHFGPLTWVCKPQGG24IA61598 GGTFSCHFGPLTWVCKPQGG12IA61894p-NO ₂ -PheGGTXSCHFGPLTWVCKPQGG17IA0.8 62019p-NO ₂ -PheGGTXSCHFGPLTWVCKPQGG18IA3.0 62020p-F-PheGGTXSCHFGPLTWVCKPQGG81.00.1 620213,5-dibromo-tyrGGTXSCHFGPLTWVCKPQGG30IAIA^* Amount required to reach maximum level 1/2 (11 pM) of EPO dependent proliferation reaction ¹ Analyzed at 10 μM ² IA = inactive ^‡ Binding to RWJ-61233 ^‡ All peptides are cyclic (except 61394) and COOH termini Analyzed as amide (-CONH ₂ ) 61233 = AFFY11157 = AFFY244
Substitution series at 17 position SEQ ID NO:orderRelative Coupling ^** EPO-ED ₅₀ (μM) Rat receptorCleaved human receptorHuman receptor 65876GGTYSCHFGPLTWVCKAQGG20 ^* 100.310^** Binding for 61233 = AFFY11157 = AFFY244 ^* limited solubility
Various active series GGTYSCHFGPLTWVCKPQGG series SEQ ID NO:orderRelative Coupling ^‡ EPO-ED ₅₀ (μM) ^*Mouse receptorsCleaved human receptor 61233 ^‡ GGTYSCHFGPLTWVCKPQGGOne0.10.1 61596GGTYSCHFGPLTWVCKPQ1.60.080.02 61718GGLYACHMGPMTWVCQPLRG0.60.10.08 AF11288GGLYACHMGPMTWVCQPLRG0.20.15ND ¹61757 (H22)LGRKYSCHFGPLTWVCQPAKKDOne0.110.15 AF11654TIAQYICYMGPETWECRPSPKAOne0.20.02 62145GGTYSCHFGPLTFVCKPQGG60.250.5 62146Ac-GGTYSCHFGPLTWVCKPQGG40.030.06 61894GGTXSCHFGPLTWVCKPQGGp-NO ₂ -Phe17IA ² 0.8 62019GGTXSCHFGPLTWVCKPQGGp-NH ₂ -Phe18IA3.0 62020GGTXSCHFGPLTWVCKPQGGp-F-Phe81.00.1^* Amount required to reach maximum level 1/2 (11 pM) of EPO dependent proliferation reaction ¹ Analyzed at 10 μM ² IA = inactive ^‡ Binding to RWJ-61233 ^‡ All peptides are cyclic (except 61394) and COOH termini Analyzed as amide (-CONH ₂ ) 61233 = AFFY11157 = AFFY244
J. Biological Analysis of Rats with Reduced Erythrocytic Hypoxia
Female BDF1 mice (18-20 g) were subjected to cycle (low pressure) treatment for 6 hours at ambient pressure for 14 days and 18 hours at 0.40 ± 0.02 atm.
Mice were kept at ambient pressure for 72 hours prior to administering γ-HuEPO or a test sample. Test samples or standard γ-HuEPO were injected into the mice receiving the treatment. This injection medium consists of PBS containing 0.1% BSA (w / v) next.
For dilution, 0.5 ml is administered subcutaneously to each of 10 rats. Blood samples are collected 48 hours after ⁵⁹ Fe administration. Erythrocyte volumetric and radiation utilization were measured for each sample. Dose ranges and results (two different assays) are described in Table 23 and FIGS. 17A-17C.
dosagenAverage log EPO St.0.000100.260.025101.650.050102.740.100103.28 DMSO0.40%70.580.35%50.60 244-10.25mg70.620.5080.761.0090.92 244-22.0091.384.00101.818.0092.26 244-314.0102.4428.0103.1556.093.16 0.1mg AFFY244 1 U r-HuEPO (8.3mg)
In addition, the biological assays of mice with reduced erythropoietic hypoxia were used to test GGTYSCHFGPLTWVCKPQGG (CAF12080) dimeric peptide analogs containing TIAQYICYMGPETWECRPSPKA and two disulfide bonds. Biological assays of mice with reduced erythropoietic hypoxia are shown in FIGS. 18A-18B. Table 24 describes suitable equivalents of EPO and various similar peptides.
PeptideAmount of equivalent peptide of 0.025 EPO (nmol) GGTYSCHFGPLTWVCKPQGG (RWJ 61233 / AFFY11157)3.8 GGTYSCHFGPLTWVCKPQ (RWJ 61596)0.5 LGRKYSCHFGPLTWVCQPAKKD (RWJ 61757)3.1 TIAQYICYMGPETWECRPSPK (AAFY11654)11-21 GGTYSCHFGPLTWVCKPQGG Dimer (AFFY12080)0.035
K. Reticulocyte Analysis
Normal untreated female BDF1 mice were injected subcutaneously with 0.5 ml for 3 consecutive days with EPO or test compound. The medium contained PBS, 10% PEG 8000, 0.25% BSA, DMSO. On day 3, iron dextran (100 mg / ml) was taken intraperitoneally by 0.1 ml. On day 5, mice were anesthetized with Co ₂ and punctured the heart to bleed. Reticulocytes (%) were determined by thiazole orange staining and flow cytometry analysis (RBC count program). Corrected reticulocytes (%) were measured according to the following formula.
RETIC (%) _{(correction value)} = RETIC _{(observation value)} × Hct _(object) / Hct _(normal)
The results obtained using the reticulocyte assay described above are shown in FIGS. 18A-18B and 19A-19B.
L. Colony Analysis: Erythrocyte Colony Formation
1. Materials and Methods
Human nucleated bone marrow cells obtained from normal donors were plated in semi-solid methylcellulose (0.9% methylcellulose, 30% fetal bovine serum, 1% bovine serum albumin). That is, cells were harvested and lysed red blood cells were lysed by adding buffered ammonium chloride. The cells were resuspended in medium containing 2% fetal calf serum and plated at 2 × 10 ⁵ cell density per two plates. Red blood cells and granulocyte-macrophage (GM) colonies formed 12 days after culture were counted based on color and morphology. Control plates containing recombinant human growth factor IL-3, GM-CSF, SCF (3 / GM / S) were included in this study to assess GM colony formation in the samples. The results are shown in Table 25.
Growth factorColony count of 2 x 10 ⁵ nucleated cells Red blood cellsG-M Reputation 1Reputation 2Reputation 1Reputation 2 Control group (no growth factor)0000 EPO (0.24)113106129 RWJ 61233 (1000)2923One2 RWJ 61233 (10,000)807520 3 / GM / S (0.7 / 0.7 / 2.7)00176212
2. Results
From the data, GGTYSCHFGPLTWVCKPQGG (RWJ 61233 / AFFY11157) can promote the formation of red blood cell colonies without additional cytokines. In addition, peptides exhibit strict specificity in the erythroid system, whereas they do not promote GM colony formation.
The foregoing is for purposes of illustration and not limitation. Those skilled in the art will be able to contemplate more embodiments as they review the specification. Accordingly, the scope of the present invention should not be limited to the above description but should be determined to be within the scope of the appended claims and equivalents. On the other hand, all references, including all patent publications herein, are described for reference.
EPO-R is useful for treating patients with low or deficient EPO deficiency or red blood cell population.

权利要求:
Claims (30)
[1" claim-type="Currently amended] Binds to an EPO (erythropoietin) receptor and contains from 10 to 40 amino acids in length containing X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ amino acid sequences, wherein each amino acid is represented by standard abbreviations X ₆ is each selected from any of 20 genetically encoded L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C peptide.
[2" claim-type="Currently amended] The amino acid sequence of claim 1, wherein the amino acid sequence contains YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ wherein each amino acid is indicated by standard abbreviation, wherein X ₂ and X ₆ are genetically encoded. Each selected from any one of twenty L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C peptide.
[3" claim-type="Currently amended] The method of claim 2 _{wherein, X 1 YX 2 X 3 X} 4 X 5 GPX 6 TWX 7 X 8 X 9 X 10 X 11 , and containing an amino acid sequence, wherein each amino acid is as indicated by the standard abbreviations method X _1, X ₂ , X ₆ , X ₉ , X ₁₀ and X ₁₁ are each selected from any one of 20 genetically encoded L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C peptide.
[4" claim-type="Currently amended] The compound of claim 3, wherein X ₄ is R, or H; X ₅ is M, or F; X ₆ is I, L, T, M, E or V; X ₇ is D or V; X ₉ is G, K, L, Q, R, S or T; X ₁₀ is a peptide A, G, P, R or Y
[5" claim-type="Currently amended] The compound of claim 4, wherein X ₁ is D, E, L, N, S, T or V; X ₂ is A, H, K, L, M, S or T; X ₄ is R or H; X ₉ is K, R, S or T; X ₁₀ is P is a peptide.
[6" claim-type="Currently amended] The peptide according to claim 1, which is selected from the following group.


[7" claim-type="Currently amended] The peptide according to claim 1, which is selected from the following group.

[8" claim-type="Currently amended] The peptide of claim 1, wherein said amino acid sequence is cyclized.
[9" claim-type="Currently amended] The peptide according to claim 8, which is selected from the following group.


[10" claim-type="Currently amended] The peptide of claim 1, wherein said amino acid sequence is dimerized.
[11" claim-type="Currently amended] The peptide of claim 10, wherein the peptide contains the following amino acid sequence:

[12" claim-type="Currently amended] A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable salt.
[13" claim-type="Currently amended] A method of treating a patient having a condition with low or defective EPO deficiency or red blood cell count, comprising administering to the patient an effective amount of the peptide of claim 1.
[14" claim-type="Currently amended] The method of claim 13, wherein the disease is terminal renal failure or dialysis; Anemia associated with AIDS, autoimmune or malignant diseases: beta-thalassemia; Cystic fibrosis; Early anemia due to immature; Anemia associated with chronic inflammatory diseases; Spinal cord injury; Acute blood loss; Aging; A tumorous disease caused by abnormal red blood cell production.
[15" claim-type="Currently amended] 14. The method of claim _{13, X 1 YX 2 X 3 X} 4 X 5 GPX 6 TWX 7 X 8 , and containing an amino acid sequence, as wherein each amino acid is indicated by standard abbreviations method, X ₂ or X ₆ password genetically engineered Selected from any one of 20 L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C.
[16" claim-type="Currently amended] The compound of claim 15, wherein the amino acid sequence contains X ₁ YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ X ₉ X ₁₀ X ₁₁ , wherein each amino acid is represented by standard abbreviation X ₁ , X ₂ , X ₆ , X ₉ , X ₁₀ and X ₁₁ are each selected from any one of 20 genetically encoded L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C
[17" claim-type="Currently amended] The method of claim 16. X ₄ is R or H; X ₅ is M or F; X ₆ is I, L, T, M or V; X ₇ is D or V; X ₉ is G, K, L, Q, R, S or T; X ₁₀ is A, G, P, R or Y.
[18" claim-type="Currently amended] The compound of claim 17, wherein X ₁ is D, E, L, N, S, T or V; X ₂ is A, H, K, L, M, S or T; X ₄ is R or H; X ₉ is K, R, S or T; X ₁₀ is P.
[19" claim-type="Currently amended] The peptide according to claim 13, which is selected from the group

[20" claim-type="Currently amended] The method of claim 13, wherein the peptide is cyclized.
[21" claim-type="Currently amended] The method of claim 13, wherein the peptide is dimerized.
[22" claim-type="Currently amended] Binds to the EPO receptor and contains the amino acid sequence X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ , wherein each amino acid is indicated by standard abbreviation, wherein X ₆ is selected from the 20 genetically encoded L-amino acids Each selected from any one; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is a method of treating a patient with a low or deficient EPO deficiency or red blood cell population, comprising administering to the patient a peptide effective amount of a 10-40 amino acid residue that is C ₈ .
[23" claim-type="Currently amended] The method of claim 22, wherein the disease is terminal renal failure or dialysis; Anemia associated with AIDS, autoimmune or malignant diseases: beta-thalassemia; Cystic fibrosis; Early anemia due to immature; Anemia associated with chronic inflammatory diseases; Spinal cord injury; Acute blood loss; Aging; A tumorous disease caused by abnormal red blood cell production.
[24" claim-type="Currently amended] The amino acid sequence of claim 22, wherein the amino acid sequence contains YX ₂ X ₃ X ₄ X ₅ GPX ₆ TWX ₇ X ₈ wherein each amino acid is indicated by standard abbreviation, wherein X ₂ and X ₆ are genetically encoded. Each selected from any one of twenty L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C.
[25" claim-type="Currently amended] 25. The method of claim _{24, X 1 YX 2 X 3 X} 4 X 5 GPX 6 TWX 7 X 8 X 9 X 10 X 11 , and containing an amino acid sequence, wherein each amino acid is as indicated by the standard abbreviations method X _1, X ₂ , X ₆ , X ₉ , X ₁₀ and X ₁₁ are each selected from any one of 20 genetically encoded L-amino acids; X ₃ is C; X ₄ is R, H, L or W; X ₅ is M, F or I; X ₇ is D, E, I, L or V; X ₈ is C.
[26" claim-type="Currently amended] The compound of claim 25, wherein X ₄ is R, H, L or W; X ₅ is M or F; X ₆ is I, L, T, M or V; X ₇ is D or V; X ₉ is G, K, L, Q, R, S or T; X ₁₀ is A, G, P, R or Y.
[27" claim-type="Currently amended] The compound of claim 26, wherein X ₁ is D, E, L, N, S, T or V; X ₂ is A, H, K, L, M, S or T; X ₄ is R or H; X ₉ is K, R, S or T; X ₁₀ is P.
[28" claim-type="Currently amended] The method of claim 22, wherein the peptide is selected from the group:

[29" claim-type="Currently amended] The method of claim 22, wherein the peptide is cyclized.
[30" claim-type="Currently amended] The method of claim 22, wherein the peptide is dimerized.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1995-06-07|Priority to US08/484635

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1995-06-07|Priority to US08/484,631

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1995-06-07|Priority to US08/484631

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1995-06-07|Priority to US8/484635

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1995-06-07|Priority to US8/484631

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1995-06-07|Priority to US08/484,635

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1996-06-07|Application filed by 린골드 고돈, 아피맥스 테크놀로지스, 엔.브이., 존 더블유. 하버, 오르토 파마슈티칼 코오포레이숀

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1999-03-25|Publication of KR19990022656A

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2005-06-20|Application granted

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2005-06-20|Publication of KR100459984B1

优先权:

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申请号 | 申请日 | 专利标题

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US08/484631|1995-06-07|

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US8/484635|1995-06-07|

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US8/484631|1995-06-07|

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US08/484,635|US5773569A|1993-11-19|1995-06-07|Compounds and peptides that bind to the erythropoietin receptor|

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US08/484635|1995-06-07|

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US08/484,631|US5830851A|1993-11-19|1995-06-07|Methods of administering peptides that bind to the erythropoietin receptor|