+

WO2006001770A1 - Methode permettant d'identifier des modulateurs des recepteurs de classe i de la cytokine - Google Patents

Methode permettant d'identifier des modulateurs des recepteurs de classe i de la cytokine Download PDF

Info

Publication number
WO2006001770A1
WO2006001770A1 PCT/SE2005/001001 SE2005001001W WO2006001770A1 WO 2006001770 A1 WO2006001770 A1 WO 2006001770A1 SE 2005001001 W SE2005001001 W SE 2005001001W WO 2006001770 A1 WO2006001770 A1 WO 2006001770A1
Authority
WO
WIPO (PCT)
Prior art keywords
growth hormone
receptor
variant
seq
binding
Prior art date
Application number
PCT/SE2005/001001
Other languages
English (en)
Inventor
Joanna Chmielewska
Susanne GRÄSLUND
Agneta Tjernberg
Original Assignee
Biovitrum Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE0401666A external-priority patent/SE0401666D0/xx
Application filed by Biovitrum Ab filed Critical Biovitrum Ab
Publication of WO2006001770A1 publication Critical patent/WO2006001770A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

  • the present invention relates to methods for the identification of modulators of cytokine class I receptors, in particular the growth hormone receptor.
  • the modulators are useful as pharmaceutical compounds, e.g., in the treatment of acromegaly.
  • the invention also provides variants of the growth hormone receptor, said variants essentially lacking capacity to bind human growth hormone.
  • GHR The Growth Hormone Receptor belongs to the cytokine class I receptor family, together with, e.g., the prolactin receptor (PRLR), the erythropoietin receptor (EPOR), the thrombopoietin receptor (TPOR) [I].
  • PRLR prolactin receptor
  • EPOR erythropoietin receptor
  • TPOR thrombopoietin receptor
  • GHR is expressed in several tissues, e.g., liver, pancreas, muscles, adipose tissue, bone and brain, where it is activated by its natural ligand, growth hormone (GH). Activation of the receptor triggers multiple signal transduction pathways, resulting in tissue / organ dependent responses known to be induced by GH [2].
  • the cDNA for human growth hormone receptor encodes a 620-aa protein of about 70 kDa (positions 19-638 of SEQ ID NO:2), consisting of an extracellular ligand binding domain, a membrane spanning region, and a cytoplasmic part, involved in signaling [3, 4].
  • growth hormone binding protein, GHbp comprising the extracellular part of the receptor, can be isolated from plasma [5].
  • GHbp is generated by alternative splicing in rodents and by proteolytic cleavage of the extracellular part of receptor in humans [6]. Signaling is initiated by a two-step dimerization process.
  • a high affinity binding of a GH molecule to one GHR is taking place, followed by the recruitment of a second GHR molecule to this complex, thus forming a signaling- productive ternary complex [7].
  • the dimerization of GHR also occurs spontaneously, independently of GH molecule, as described recently by Waters et al. [8]. According to this hypothesis, the GHR occurs as already predimerized form on the cellular surface. Binding of GH to these predimerized receptors introduces the conformational change in the intracellular part of receptors proximal to the cellular membrane [8]. According to Waters, it is this conformational change and not dimerization process per se, which causes initiation of cellular signaling.
  • Fig. 4 is a mass spectrogram depicting interaction between GHbpwi 04A and compound BVT.3693.
  • Fig. 5 is a mass spectrogram depicting interaction between GHbp 13 o -237 ([M], left panel) or GHbp 32-237 ([M], right panel), compound BVT.3693 [L], and a competing ligand BVT.39221 [R].
  • the said assay in step (ii) is a competitive binding assay comprising the steps: (a) contacting the growth hormone receptor or a variant thereof with a test compound under conditions that permit binding to the growth hormone receptor or variant thereof; and (b) determining whether the test compound binds to the growth hormone receptor or variant thereof by detecting the presence or absence of a signal generated from the interaction of the test compound with the growth hormone receptor or variant thereof, in the presence of the molecule provided in step (i).
  • binding of test compounds to the GH receptor was determined using Electrospray-Ionization Mass Spectrometry (ESI-MS) technology.
  • ESI-MS Electrospray-Ionization Mass Spectrometry
  • the molecule provided in step (i) could be labeled and its displacement from the receptor by a competing compound can be monitored by known methods such as SPA (Scintillation Proximity Assay) technology.
  • SPA Scintillation Proximity Assay
  • a FRET assay can be employed.
  • the said competitive binding assay can preferably be carried out using a variant of the said growth hormone receptor, said variant lacking capacity to bind growth hormone.
  • the said variant could, e.g., have a point mutation inhibiting its capacity to bind growth hormone.
  • variants are derived from the extracellular polypeptide GHbp 32-237 (SEQ ID NO:3) corresponding to positions 50-255 of SEQ ID NO:2.
  • variants can be obtained from other extracellular polypeptides (GH binding proteins) derived from GHR shown as SEQ ID NO:2.
  • extracellular polypeptides include polypeptides referred to as GHbpi. 237 (positions 19-255 of SEQ ID NO:2); GHbp 1-246 (positions 19-264 of SEQ ID NO:2); and GHbp 32-24 6 (positions 50-264 of SEQ ID NO:2).
  • the said molecule identified or provided in step (i) above could, e.g., be a peptide, as exemplified by the peptide having the amino acid sequence of SEQ ID NO:6.
  • the molecule is a low-molecular weight molecule such as the compound designated BVT.3693 (N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2- furamide).
  • BVT.3693 N-[5-(aminosulfonyl)-2-methylphenyl]-5-bromo-2- furamide.
  • the present invention provides a compound identified as an agonist or an antagonist of GHR for use in therapy, in particular for treating acromegaly, cancer, diabetes, diabetic nephropathy, diabetic retinopathy and neuropathy, and other diseases with pathologically increased IGF-I levels, as well as for treatment of children with growth hormone deficiency, Prader- Willis syndrome, Turners syndrome, children with retarded growth due to chronic renal failure, substitution of adults with growth hormone deficiency, frail elderly, and wasting syndrome in AIDS.
  • the invention provides a method for the identification of a modulator of a cytokine class I receptor, comprising: (i) identifying or providing a molecule that binds to a site at the cytokine class I receptor different from the binding site of the natural ligand of the cytokine class I receptor; (ii) using the molecule in a competitive binding assay for identifying a modulator to the cytokine class I receptor, wherein the competitive binding assay is carried out using a variant of the cytokine class I receptor that does not bind to the natural ligand of the cytokine class I receptor.
  • cytokine class I receptor is well known in the art.
  • the family of cytokine class I receptors comprises, e.g., the growth hormone receptor (GHR), the prolactin receptor (PRLR), the erythropoietin receptor (EPOR), and the thromobopoietin receptor (TPOR) [I].
  • the invention provides a polypeptide which is a variant of the polypeptide having the amino acid sequence shown as SEQ ID NO:3, but essentially lacking capacity to bind growth hormone.
  • the said variant could, e.g., have a point mutation inhibiting its capacity to bind growth hormone, such as the variant GHbp 32-237 wi04A (SEQ ID NO:4), or lack the growth hormone binding region, such as the variant GHbpi 30-237 (SEQ ID NO:5).
  • the said variant should preferably retain the ability to interact in the extracellular stem region of GHR with a second GHR molecule in a dimerization process; and should preferably have an intact domain 2 of the extracellular part of the receptor.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • a variant GH binding protein (GHbp 32- 2 37 wi04A; SEQ ID NO:4) was unable to bind GH due to a point mutation at position 104.
  • a circular DNA consisting of 5061 base pairs and coding for the same binding protein, but with TrplO4 replaced with Ala, and with the addition of a Hisg-tag sequence at the C-terminus, was prepared.
  • the W 104 A mutation was inserted using oligonucleotides containing the new codon in a two-step Polymerase Chain Reaction method. Expression of the encoded protein was thus performed in E. coli strain BL21 in a 5 L bench-top fermenter.
  • the protein which could not be purified by a traditional GH-aff ⁇ nity chromatography, was purified to homogeneity in a three-step purification process comprising (i) osmotic chock; (ii) immobilized metal-ion affinity chromatography (IMAC) utilizing the His 6 -tag; and (iii) gel filtration.
  • the two chromatographic steps were performed on an Akta Explorer System (Amersham Biosciences) using a TALON column (Clontech) and a HiLoad X16/60 Superdex 75 (Amersham Biosciences).
  • the amount and the purity of the produced protein were analyzed by SDS-PAGE, N-terminal sequencing, amino acid analysis and MALDI-MS.
  • a further GHbp variant comprising domain 2 (amino acids 130-137) of the GH receptor, was prepared (GHbp I30-237 ; SEQ ID NO: 5).
  • domain I amino acids 130-137
  • SEQ ID NO: 5 A further GHbp variant, comprising domain 2 (amino acids 130-137) of the GH receptor.
  • the protein which could not be purified by a traditional GH-affinity chromatography, was purified to homogeneity in a three-step purification process comprising (i) Ammonium sulphate precipitation; (ii) gel filtration; and (iii) ion exchange chromatography.
  • the two chromatographic steps were performed on an Akta Explorer System (Amersham Biosciences) using a HiLoad X26/60 Superdex 75 (Amersham Biosciences) and a Mono Q HR 10/10 IEX column (Amersham Biosciences). The amount and the purity of the produced protein were analyzed by SDS-PAGE, C- and N-terminal sequencing, amino acid analysis and MALDI-MS.
  • EXAMPLE 2 ESI-MS determination of ligand-receptor binding Binding of test compounds to the GH receptor was determined using Electrospray-Ionization Mass Spectrometry (ESI-MS) technology.
  • ESI-MS Electrospray-Ionization Mass Spectrometry
  • Non-denaturing ESI- MS allows the transfer of non-covalently bound complexes from a solution into the gas phase.
  • the identification of non-covalent host-guest interactions by mass spectrometry has been known since 1991 [21, 22,23].
  • the sample in solution is sprayed directly by a strong electric field gradient into the gas phase [24,25].
  • the peak intensity of the protein-reference ligand complex was measured before and after the addition of a further ligand, competing for the same binding site on the protein. Similar procedures for determination of binding affinities has previously been used by Kempen et al. [27] for measuring binding constants of crown ethers:alkali metal complexes, and by Oliv [28] for the complexes between model proteins and known low molecular mass binders (300-500 Da). The proteins were dialyzed offline against 10 mM ammonium acetate by means of a Slide-a-lyzer prior to analysis (MWCO 10000, Pierce, Rockford, IL). The ESI mass spectra were recorded on a Q-TOF-I instrument (Micromass, Manchester, UK).
  • the instrument conditions for the ESI interface were as follows: Capillary voltage 3000 V, cone voltage 80 or 90 V, source block temperature 6O 0 C, desolvation temperature 80°C.
  • an elevated instrument pressure in the source region was maintained by reducing analyzer pumping and introducing gas into the hexapole collision cell to achieve an analyzer pressure of approximately 4xlO' 5 mbar.
  • the mass spectra were recorded between 1000 and 5000 m/z, with data accumulation of 2 s per spectrum and an inter scan time delay of 0.1 s. 65 scans/experiment were performed.
  • the mobile phase used was ammonium acetate (10 mM; pH 6.5), and the sample flow rate was 5 ⁇ l/min. A volume of 5 ⁇ l was injected by means of an autosampler (FAMOS, LC packings, Amsterdam, NL).
  • EXAMPLE 3 GHbpi?.??7 WI ⁇ UA does not form a complex with growth hormone Human GH was obtained from Pharmacia, (Genotropin®, batch 25690-51). GHbp 32-237 (6 ⁇ M) was incubated with human GH (5.5 ⁇ M) for 15 minutes prior to MS analysis. For the experiments with the W104A mutant, 10 ⁇ M of GHbp 32-237 wio4A was preincubated with 13 ⁇ M of hGH. ESI-MS was then carried out as described in Example 2. As shown in Fig. 1 (upper panel) GHbP 32-237 formed a complex with human GH, whereas the mutant GHbp 32 .
  • the 1 :1 and 1:2 complexes between GH and GHbp 32-237 are indicated as (A) and (B). Complexes formed in the absence of peptide are shown in the upper panel, and complexes formed in the presence of peptide in the lower panel. Additional peaks were detected, corresponding to the multiple charges 15 and 16 of the complex 1 :1 :1 at 49.6 kDa and to the multiple charges 18 and 19 of the complex 1 :2:1 at 73.6 kDa. This indicated that 1 :1 :1 as well as 1 :2:1 complexes were formed between GH-GHbp-peptide. In addition, the results indicate that the peptide binds to a site different from the GH-binding site on the receptor, since the peptide does not displace GH in the complex.
  • EXAMPLE 5 Screening for LMW compounds A homogenous screening assay, employing the GHR-derived peptide (SEQ ID NO: 6) as a competitive ligand, was designed based on Fluorescence Energy Transfer (FRET) principle [29]. Briefly, biotine labeled peptide and GHR specific monoclonal antibody labeled with Europium are bound to the receptor. The signaling molecule - allophycocyanin bound to streptavidin is then added to the reaction mixture, whereupon streptavidin forms a complex with a peptide-bound biotine. This interaction brings Eu and allophycocyanin in proximity, allowing for energy transfer between europium and allophycocyanin, resulting in emission signal of fluorescence at 665 run wavelength. If the peptide is displaced from the receptor by a competing ligand, a diminished signal is measured.
  • FRET Fluorescence Energy Transfer
  • the 1 : 1 complex between the substance and the protein is indicated.
  • the excess of the free protein shown in Fig. 4 is due to dissociation of the complex when the sample enters the gas phase inside the mass spectrometer. In solution, the 1 : 1 complex is probably more abundant.
  • EXAMPLE 8 Low-molecular weight compounds binds to GHbpnn-rv? Another protein lacking GH-binding domain used in the studies was GHbp 13 o- 237 . In this construct the whole domain involved in GH-interactions was deleted. Despite this, the truncated protein was able to form a 1 :1 complex with BVT.3693. A competition study was done using BVT.3693 at a fixed concentration of 30 ⁇ M. A competing ligand, 5-bromo-N-[2,4,6-trimethyl-3-(morpholin-4- ylsulfonyl)phenyl]-2-furamide (BVT.39221), was added at increasing concentrations (0-300 ⁇ M).
  • the proteins GHbPi 30-237 (left panel), or as a control GHbp 32-237 (right panel), were used at 6 ⁇ M concentrations.
  • the results (Fig. 5) indicate that both LMW compounds could form complexes with GHbp ⁇ o -23 7 to the same extent they form complexes with GHbp 32 . 237j which has an intact GH binding region. It could also be seen that both ligands compete for the same binding site on the proteins.
  • GHbp variants which lack the GH binding region, can still be used in screening assays to identify modulating molecules binding to a site at the GHR different from the GH binding site.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Endocrinology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des méthodes permettant d'identifier des modulateurs des récepteurs de classe I de la cytokine, en particulier, le récepteur de l'hormone de croissance. Ladite méthode consiste à identifier une molécule se liant au niveau du récepteur à un site différent du site de liaison du ligand naturel de ce récepteur, et à utiliser ladite molécule dans un dosage de liaison compétitive afin d'identifier des modulateurs du récepteur de classe I de la cytokine.
PCT/SE2005/001001 2004-06-28 2005-06-23 Methode permettant d'identifier des modulateurs des recepteurs de classe i de la cytokine WO2006001770A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE0401666-3 2004-06-28
SE0401666A SE0401666D0 (sv) 2004-06-28 2004-06-28 Screening methods
US60959304P 2004-09-14 2004-09-14
US60/609,593 2004-09-14

Publications (1)

Publication Number Publication Date
WO2006001770A1 true WO2006001770A1 (fr) 2006-01-05

Family

ID=35782082

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2005/001001 WO2006001770A1 (fr) 2004-06-28 2005-06-23 Methode permettant d'identifier des modulateurs des recepteurs de classe i de la cytokine

Country Status (1)

Country Link
WO (1) WO2006001770A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012123734A1 (fr) * 2011-03-15 2012-09-20 Asterion Limited Protéines de fusion de récepteur modifié
WO2016130901A2 (fr) 2015-02-12 2016-08-18 University Of Southern California Bloqueurs du récepteur de l'hormone de croissance utilisés dans la prévention et le traitement de maladies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06172394A (ja) * 1992-12-10 1994-06-21 Tanpaku Kogaku Kenkyusho:Kk 成長ホルモンレセプターの細胞質外ｃ−ドメイン蛋白質
US6333031B1 (en) * 1996-03-08 2001-12-25 Reception, Inc. Receptor derived peptides as modulators of receptor activity
US20030009009A1 (en) * 1997-02-18 2003-01-09 Michael Sundstrom Modified cytokine receptor protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06172394A (ja) * 1992-12-10 1994-06-21 Tanpaku Kogaku Kenkyusho:Kk 成長ホルモンレセプターの細胞質外ｃ−ドメイン蛋白質
US6333031B1 (en) * 1996-03-08 2001-12-25 Reception, Inc. Receptor derived peptides as modulators of receptor activity
US20030009009A1 (en) * 1997-02-18 2003-01-09 Michael Sundstrom Modified cytokine receptor protein

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 1 March 1995 (1995-03-01), Database accession no. (AAR56390) *
DATABASE EMBL [online] 1 March 1995 (1995-03-01), Database accession no. (AAR56391) *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012123734A1 (fr) * 2011-03-15 2012-09-20 Asterion Limited Protéines de fusion de récepteur modifié
US9493536B2 (en) 2011-03-15 2016-11-15 Asterion Limited Growth hormone receptor antagonists
US10030062B2 (en) 2011-03-15 2018-07-24 Asterion Limited Growth hormone fusion proteins with growth hormone receptor agonist activity
WO2016130901A2 (fr) 2015-02-12 2016-08-18 University Of Southern California Bloqueurs du récepteur de l'hormone de croissance utilisés dans la prévention et le traitement de maladies
WO2016130901A3 (fr) * 2015-02-12 2016-10-20 University Of Southern California Bloqueurs du récepteur de l'hormone de croissance utilisés dans la prévention et le traitement de maladies
CN107438605A (zh) * 2015-02-12 2017-12-05 南加利福尼亚大学 疾病预防和治疗中的生长激素受体阻断剂
JP2018506534A (ja) * 2015-02-12 2018-03-08 ユニヴァーシティ オブ サザン カリフォルニアＵｎｉｖｅｒｓｉｔｙ ｏｆ Ｓｏｕｔｈｅｒｎ Ｃａｌｉｆｏｒｎｉａ 疾患予防および治療における成長ホルモン受容体の遮断剤
US10246446B2 (en) 2015-02-12 2019-04-02 University Of Southern California Blockers of the growth hormone receptor in disease prevention and treatment
RU2726254C2 (ru) * 2015-02-12 2020-07-10 Юниверсити Оф Саутерн Калифорния Блокаторы рецептора гормона роста при предупреждении заболеваний и лечении

Similar Documents

Publication Publication Date Title
Gemel et al. Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells
Mori et al. Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor.
CN101784281B (zh) 确定g蛋白偶联受体激动剂的方法
Hsu et al. Selective deamidation of recombinant human stem cell factor during in vitro aging: isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers
CA2402392A1 (fr) Recepteurs connus couples a la proteine g non endogenes a activation constitutive
JPWO2002044362A1 (ja) 糖尿病治療剤スクリーニング方法
RU95106629A (ru) Днк, конструкция днк, клетка-хозяин, способ получения рецептора, пептид, антитело, зонд, способ обнаружения
US20020115110A1 (en) Method of identifying agonist and antagonists for tumor necrosis related receptors TR1 and TR2
CA2245924A1 (fr) Methode d'identification d'agonistes et d'antagonistes des recepteurs lies a la necrose des tumeurs tr1, tr3 et tr5
Seiffert et al. Orchestration of signaling by structural disorder in class 1 cytokine receptors
Scaloni et al. Purification, cloning and characterisation of odorant-and pheromone-binding proteins from pig nasal epithelium
Jin et al. Novel conorfamides from Conus austini venom modulate both nicotinic acetylcholine receptors and acid-sensing ion channels
Song et al. Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT
Alalwiat et al. Mass spectrometry and ion mobility characterization of bioactive peptide–synthetic polymer conjugates
Eriste et al. A novel form of neurotensin post-translationally modified by arginylation
Miranda et al. Identification of potent, selective, and metabolically stable peptide antagonists to the calcitonin gene-related peptide (CGRP) receptor
US20050191612A1 (en) Method of screening insulin content enhancer
EP1560025A3 (fr) Marquers specifiques pour diabète
Darie et al. Blue native page and mass spectrometry analysis of ephrin stimulation-dependent protein-protein interactions in Ng108-Ephb2 cells
WO2006001770A1 (fr) Methode permettant d'identifier des modulateurs des recepteurs de classe i de la cytokine
Vogel et al. EPOR-based purification and analysis of erythropoietin mimetic peptides from human urine by Cys-specific cleavage and LC/MS/MS
Miranda et al. Peptide antagonists of the calcitonin gene‐related peptide (CGRP) receptor with improved pharmacokinetics and pharmacodynamics
Liu et al. Targeted top-down mass spectrometry for the characterization and tissue-specific functional discovery of crustacean hyperglycemic hormones (CHH) and CHH precursor-related peptides in response to low pH stress
Quigley et al. Orphanin FQ is the major OFQ1–17-containing peptide produced in the rodent and monkey hypothalamus
Fischer et al. The Impact of Adrenomedullin Thr22 on Selectivity within the Calcitonin Receptor‐like Receptor/Receptor Activity‐Modifying Protein System

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载