JP4293971B2 - Fp−グレリン - Google Patents
Fp−グレリン Download PDFInfo
- Publication number
- JP4293971B2 JP4293971B2 JP2004302925A JP2004302925A JP4293971B2 JP 4293971 B2 JP4293971 B2 JP 4293971B2 JP 2004302925 A JP2004302925 A JP 2004302925A JP 2004302925 A JP2004302925 A JP 2004302925A JP 4293971 B2 JP4293971 B2 JP 4293971B2
- Authority
- JP
- Japan
- Prior art keywords
- labeled
- growth hormone
- ghrelin
- hormone secretagogue
- acid
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired - Fee Related
Links
- 102100033367 Appetite-regulating hormone Human genes 0.000 claims description 64
- GNKDKYIHGQKHHM-RJKLHVOGSA-N ghrelin Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)CN)COC(=O)CCCCCCC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C1=CC=CC=C1 GNKDKYIHGQKHHM-RJKLHVOGSA-N 0.000 claims description 54
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- 238000013459 approach Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000011948 assay development Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 229960004365 benzoic acid Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- AFAXGSQYZLGZPG-UHFFFAOYSA-N ethanedisulfonic acid Chemical compound OS(=O)(=O)CCS(O)(=O)=O AFAXGSQYZLGZPG-UHFFFAOYSA-N 0.000 description 1
- 238000010228 ex vivo assay Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 108010070965 hexarelin Proteins 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 229940045996 isethionic acid Drugs 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- TYQCGQRIZGCHNB-JLAZNSOCSA-N l-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(O)=C(O)C1=O TYQCGQRIZGCHNB-JLAZNSOCSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- LNOPIUAQISRISI-UHFFFAOYSA-N n'-hydroxy-2-propan-2-ylsulfonylethanimidamide Chemical compound CC(C)S(=O)(=O)CC(N)=NO LNOPIUAQISRISI-UHFFFAOYSA-N 0.000 description 1
- AEMBWNDIEFEPTH-UHFFFAOYSA-N n-tert-butyl-n-ethylnitrous amide Chemical compound CCN(N=O)C(C)(C)C AEMBWNDIEFEPTH-UHFFFAOYSA-N 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 230000005803 octanoylation Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229940066842 petrolatum Drugs 0.000 description 1
- 235000019271 petrolatum Nutrition 0.000 description 1
- BSCCSDNZEIHXOK-UHFFFAOYSA-N phenyl carbamate Chemical class NC(=O)OC1=CC=CC=C1 BSCCSDNZEIHXOK-UHFFFAOYSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Images
Classifications
-
- 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
- C07K14/575—Hormones
- C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
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- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
R1-Cys-F
の標識された成長ホルモン分泌促進物質に関し、式中、R1はグレリン(ghrelin)のポリペプチド配列(配列番号:1)に由来するペプチド配列であり、Fは蛍光色素である。1つの好ましい態様において、R1は配列番号:2である。もう1つの好ましい態様において、R1はオクタノイル化(octanoylated)されている。もう1つの好ましい態様において、R1はN-オクタノイル化されている。より好ましい1つの態様において、R1は
である。N-オクタノイル化はエステラーゼ活性に対するグレリンの安定性を高める。さらにより好ましい1つの態様において、Fはテキサスレッド、テトラメチルローダミン、MR121からなる群より選択される(「New fluorescent dyes in the red region for biodiagnostics」、M. Sauerら、1995 J. Fluoresc. Vol. 5、pp 247-261、またはBODIPY-FL 4,4-ジフルオロ-5,7-ジメチル-4-ボラ-3a,4a-ジアザ-s-インダセン-3-プロピオン酸)。最も好ましい1つの態様において、FはMR121である。
R1-Cys-F
(式中、R1はグレリンのポリペプチド配列に由来するペプチド配列であり、Fは蛍光色素である)
a)CysをC末端アミノ酸と結合させる段階;および
b)そのチオール含有グレリンを蛍光色素と反応させる段階、
を含む工程であることを特徴とする。
a)前記化合物を、成長ホルモン分泌促進物質受容体を発現する宿主から単離された膜と、アッセイ緩衝液中にて、上記(1)〜(7)のいずれか一項記載の標識された成長ホルモン分泌促進物質の存在下で、接触させる段階;
b)結合していない標識された成長ホルモン分泌促進物質を除去するために前記膜を洗浄する段階;および
c)化合物が、上記(1)〜(7)のいずれか一項記載の標識された成長ホルモン分泌促進物質と成長ホルモン分泌促進物質受容体との結合に影響を及ぼすか否かを、前記膜の蛍光を測定することによってモニタリングする段階、
を含む方法であることを特徴とする。
実施例1:標識ペプチドの合成
実施例1.1:
の合成
「Fmoc Solid Phase Peptide Synthesis:A practical approach」(Oxford University Press 2000)pp 41-74、W.C. ChanおよびP.D. White編に記載された方法に従い、Tenta Gel S RAM樹脂(0.25mmol/g(Rapp Polymere GmbH, Tubingen, Germany)を出発材料として、Pioneer(商標)ペプチド合成システムにて連続流固相合成を実施した。塩基不安定性のあるFmoc基をα-アミノの保護に用いた。側鎖は以下の保護基により保護した:-Asn(Trt)、Glu(OtBu)、Ser(tBu)、His(Trt)、Gln(Trt)、His(Trt)、Arg(Pbf)、Lys(Boc)、Cys(Trt)。Fmoc-アミノ酸(4当量)を1当量のテトラフルオロホウ酸O-(1,2-ジヒドロ-2-オキソピリド-1-イル)-N,N,N',N'-テトラメチルウロニウム(TPTU)およびN,N-ジイソプロピルエチルアミン(ヒューニッヒ(Hunig)塩基)により活性化した。Fmocの脱保護は20%ピペリジンを含むDMFにより行った。残基4Pheが標的配列に組み込まれた後に自動合成を中止した。Peptide Synthesizer SP650(Labortec AG)を用いてペプチド合成を半手動的に続けた。側鎖が保護されていないFmoc-3Ser-OH(0.65g、2mmol)、TPTU(0.59g、2mmol)、ヒューニッヒ塩基(1.03ml)をペプチド樹脂に添加し、DMF溶媒(ニンヒドリン陰性)中で結合反応を1時間継続させた。側鎖ヒドロキシルのオクタノイル化は、N-メチルピロリジン溶媒中にてカプリル酸(2.0ml、12mmol)、N,N'-ジイソプロピルカルボジイミド(1.9ml、12mmol)、N,N'-ジメチルアミノピリジン(18mg、0.15mmol)を用いて行った。4時間後に反応混合物を濾過し、標準的なペプチド合成プロトコール(上記)を用いて合成を続けた。
Tenta Gel S-樹脂(2.0g)を、95%TFA、2.5%H2O、2.5%EDT、2.5%トリイソプロピルシランからなる混合物(100ml)により5時間処理した。この反応混合物を濃縮して、ジエチルエーテルに注ぎ入れ、沈殿物を濾過によって収集した上で、凍結乾燥させて水分を除去した。粗ペプチド(0.80g)を調製用RP-HPLCにより精製した。
が得られた(0.18g、イオンスプレーMS分析(M+2H)2+/2=1165.4、(M+3H)3+/3=777.2)。
の合成
(S)-2-(9H-フルオレン-9-イルメトキシカルボニルアミノ)-3-オクタノイルアミノ-プロピオン酸;Fmoc-L-Dapa(N-オクタノイル)-OH
DMF(20ml)中にカプリン酸(1.54ml、10mmol)、TPTU(2.8g、9.5mmol)、およびヒューニッヒ塩基(3.4ml、20mmol))を含むあらかじめ活性化された混合物に対して、Fmoc-L-Dapa-OH Neosysytem FA040028(3.3g、10mmol)を含むDMF溶液(10ml)を添加した。この反応混合物を1時間攪拌し、減圧下で濃縮した上で酢酸エチル中に溶解し、5%/10%KHSO4/K2SO4で洗浄し、塩水に浸し、Na2SO4で乾燥させ、濾過した上で濃縮した。酢酸エチル/ヘキサンによる結晶化:3.7g、82%;MS=451.4(MH)−。
を得た:135mg;イオンスプレーMS:(M+2H)2+/2=1164.8,(M+3H)3+/3=776.8)。
実施例1.2:
上記のチオール含有ペプチド(1.3mg)を9:1 DMSO:50mMリン酸緩衝液pH 6中に最終濃度2.5mMとして溶解した。1.2当量のTxR(テキサスレッド)-マレイミド(DMSO中に新たに調製した30mM溶液)を添加し、混合物を室温で10分間反応させた。この反応混合物をRP-HPLCにより直接精製した:0.17mg;MS分析:算出されたモノアイソトピック質量:C136H198N36O39S3=3055.38;観測されたモノアイソトピック質量:3055.35。
TMR(テトラメチルローダミン)により誘導体化したペプチドを、実施例1.2と同じように調製した:1.7mgのペプチド出発材料から0.44mgの標識ペプチドが単離された。MS分析:算出されたモノアイソトピック質量:C127H185N35O36S=2808.34;観測されたモノアイソトピック質量:2808.40。
MR121*により誘導体化したペプチドを実施例1.2と同じように調製した。1.5mgの最初のペプチド材料から0.37mgの標識ペプチドが単離された。MS分析:算出されたモノアイソトピック質量:C129H196N37O35S=2855.44;観測されたモノアイソトピック質量:2855.38。
BODIPY-FL(4,4-ジフルオロ-5,7-ジメチル-4-ボラ-3a,4a-ジアザ-s-インダセン-3-プロピオン酸)により誘導体化したペプチドを実施例1.2と同じように調製した:0.7mgの出発材料から0.20mgの標識ペプチドが単離された。MS分析:算出されたモノアイソトピック質量:C119H183BF2N36O34S=2742.4;観測されたモノアイソトピック質量:2742.4。
MR121*により誘導体化したペプチドを実施例1.2と同じように調製した:0.7mgの最初のペプチド材料から0.17mgの標識ペプチドが単離された。MS分析:算出されたモノアイソトピック質量:C129H197N36O35S=2854.46;観測されたモノアイソトピック質量:2854.49。
実施例2.1:膜の調製
ヒト胎児腎臓HEK 293(EBNA)細胞を懸濁液中で増殖させ、以前に記載された方法に従ってトランスフェクションを行った(SchlaegerおよびChristensen、Cytotechnology, 30, 71-83, 1999)。細胞を500rpmで10分間遠心し、PBS-0.7mM EDTA/(4℃)で1回洗った後に、PBS-EDTA-PI(プロテアーゼ阻害剤混合物を含む)中に2ml/細胞1gとして再懸濁した。細胞をUltra Turax(グリーンレベル)により15''×3回、氷上で30''破砕した。細胞片を除去するために懸濁液をSorvall SS34ローターにより2'000rpmで20分間遠心した。上清を採取し、さらに20'000rpmで40分間遠心した。ペレットをPBS-EDTA中に再懸濁した。T-MK 0677を用いた飽和結合アッセイ法により、受容体密度は4.9pmol/mgタンパク質であることが確かめられた。
実施例2.2.1 アッセイ法の開発
GHSR-1aを含む細胞膜をFP-緩衝液:25mM Hepes、5mM MgCl2、1mM CaCl2、4%PEG、0.1%BSA(第V画分)中に最終容積が0.5〜1mlとなるように希釈し、0.4mmシリンジを通過させた上で、氷上に保ちながら20パルスによる超音波処理を4回行った。MR121-標識グレリン(トレーサー)の10nM溶液、および競合物質を含むFP-緩衝液を20倍に濃縮した溶液を調製した。受容体に結合したトレーサーの偏光を測定するために、180μlの試料を3種類調製した:全結合:膜+トレーサー、遊離リガンド:膜+トレーサー+競合物質、蛍光バックグラウンド:膜+緩衝液。3×50μlの各試料を混合した後、直ちにアッセイ用プレートに移した。
図1は、ハイスループットスクリーニング用の384ウェルプレートに関して考えられるレイアウトを示している。
1)1.33×膜溶液30μlをアッセイ用プレートのすべてのウェルに添加した。
2)緩衝液4.4μlを「FPBLK」/「100%対照」ウェルに移し、基準化合物溶液4.4μlを「STD」ウェルおよび「0%対照」ウェルに、蓄積用プレートからアッセイ用プレートに移した。
3)水10μl(0%DMSO)を、1μlの2mM化合物(最終濃度20μM)を含む化合物貯蔵プレートのカラム3〜24に添加した。
4)貯蔵プレートの内容物を混合した。
5)希釈した化合物4.4μlを貯蔵プレートからアッセイ用プレートに移した。
6)アッセイ用プレートの内容物を5回混合した後に24℃で30分間インキュベートした。
7)7.143×トレーサー溶液5.6μlをアッセイ用プレートに添加した。ただし、ウェルA1〜D2(図1で「FPBLK」と表記したウェル)には代わりに緩衝液5.6μlを添加した。
8)アッセイ用プレートの内容物を5回混合した。
9)各ウェル中の溶液30μlをアッセイ用プレートから読取り用プレート(Corning UV非結合性表面)に移した。
10)読取り用プレートを室温で10分間インキュベートした。
11)Zeiss plate::visionマイクロタイタープレートリーダーを用い、平行(‖)および直交(⊥)偏光の設定で、650〜695nmのMR121蛍光を読取り用プレートから読み取った(5秒間、底面に対して1mmに焦点、xyスキャン0.5mm)。
Claims (12)
- 下記式の標識グレリン:
R1-Cys-F
(式中、R1は配列番号2のグレリンのポリペプチド配列であり、CysはR1のC末端のアミノ酸に結合しており、Fは蛍光色素である) - R1がオクタノイル化されている、請求項1記載の標識グレリン。
- R1がN-オクタノイル化されている、請求項1記載の標識グレリン。
- FがTxR、TMR、MR121、またはBODIPY-FLからなる群より選択される、請求項1〜5のいずれか一項記載の標識グレリン。
- 成長ホルモン分泌促進物質受容体と結合しうる化合物を同定するための、請求項1〜6のいずれか一項記載の標識グレリンの使用。
- 細胞受容体を成長ホルモン分泌促進物質受容体として同定するための、請求項1〜6のいずれか一項記載の標識グレリンの使用。
- 請求項1〜6の標識グレリンを合成する工程であって、
a)Cysを配列番号:2のポリペプチドのC末端アミノ酸と結合させる段階;および
b)そのチオール含有グレリンを蛍光色素と反応させる段階、
を含む工程。 - 成長ホルモン分泌促進物質受容体と結合しうる化合物を同定するための方法であって、
a)該化合物を、成長ホルモン分泌促進物質受容体を発現する宿主から単離された膜と、アッセイ緩衝液中にて、請求項1〜6のいずれか一項記載の標識された成長ホルモン分泌促進物質の存在下で、接触させる段階;
b)結合していない標識された成長ホルモン分泌促進物質を除去するために該膜を洗浄する段階;および
c)化合物が、請求項1〜6のいずれか一項記載の標識された成長ホルモン分泌促進物質と成長ホルモン分泌促進物質受容体との結合に影響を及ぼすか否かを、該膜の蛍光を測定することによってモニタリングする段階、
を含む方法。 - ハイスループットスクリーニング方法である、請求項10記載の方法。
- アッセイ緩衝液のpHが7.2である、請求項10または11記載の方法。
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RU2459831C2 (ru) * | 2006-09-27 | 2012-08-27 | Ипсен Фарма С.А.С. | Аналоги грелина с замещением на n-конце |
WO2009140763A1 (en) * | 2008-05-23 | 2009-11-26 | The University Of Western Ontario | Novel ghrelin analogues |
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US7186798B2 (en) | 2007-03-06 |
CA2480199A1 (en) | 2005-04-16 |
DE602004012207T2 (de) | 2009-03-12 |
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