JP6675119B2 - 選択的な染色体タンパク質のアシル化を行うための人工触媒システム - Google Patents
選択的な染色体タンパク質のアシル化を行うための人工触媒システム Download PDFInfo
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- JP6675119B2 JP6675119B2 JP2015169448A JP2015169448A JP6675119B2 JP 6675119 B2 JP6675119 B2 JP 6675119B2 JP 2015169448 A JP2015169448 A JP 2015169448A JP 2015169448 A JP2015169448 A JP 2015169448A JP 6675119 B2 JP6675119 B2 JP 6675119B2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
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Description
(2)(1)に記載の化合物とアシルCoA若しくはその誘導体との組み合わせを含む、染色体タンパク質をアシル化するための薬剤。
本発明は、標的アシル化領域結合性触媒、具体的には、下記の構造を有する化合物を提供する。
本発明の標的アシル化領域結合性触媒は、本発明の人工触媒システムの一つの構成要素であり、アシルCoA若しくはその誘導体との組み合わせで、高い選択性でタンパク質のアシル化を行うことができる。
本実施例において、標的アシル化領域結合性触媒とアシルCoA若しくはその誘導体との組み合わせにより、染色体タンパク質をアシル化できることが見出された。従って、本発明は、上記標的アシル化領域結合性触媒とアシルCoA若しくはその誘導体との組み合わせを含む、染色体タンパク質をアシル化するための薬剤を提供する。また、本発明は、当該組み合わせを用いる、染色体タンパク質をアシル化する方法を提供する。
1.細胞分画
約106個の細胞をトリプシン処理により培養ディッシュより剥がし、PBSで洗浄後、細胞ペレットを冷却したcell lysis buffer [50mM Tris(pH7.5), 300mM NaCl, 0.3% Triton X-100, protease inhibitor cocktail および1mM PMSF]で懸濁し、30分氷上に静置した。遠心(4℃, 1500rpmで2分間)後、上清を細胞質画分として回収した。
タンパク質を4-20% SDS-PAGEゲルで分離し、PVDF膜に転写した後、TBSTに懸濁した5%スキムミルクによりブロッキングを行い、PVDF膜に1次抗体を反応させた。用いた1次抗体は以下の通りである。
緩衝液(50mM HEPES, 150mM NaCl, 0.01% Triton, pH7.5)に細胞質画分(20%)とアセチル基供与体(1mM)を加え、室温で10時間反応させた後、抗アセチルリジン抗体を用いてウエスタンブロッティングを行った。
再構成ヌクレオソーム(33μg/mL 601 DNA)および細胞質画分(15%)を含む緩衝液(20mM Tris-HCl, pH7.5)に、Ligand-DSH(2〜5μM), アシルCoA(1mM), TCEP(100μM)を加え、室温で5時間反応させた後、ウエスタンブロッティングを行った。
(1)サンプル調製
Ligand-DSH(2μM), アセチルCoA(1mM), TCEP(100μM)を緩衝液(20mM Tris-HCl, pH7.5)中で1時間反応させた後、再構成ヌクレオソーム(33μg/mL 601 DNA)を加えてさらに5時間反応させた(最終液量150μL)。冷却したトリクロロ酢酸(30uL)を加えて、氷上で30分間静置し、遠心後(4℃, 15000rpmで5分間)上清を捨てた。さらに遠心(4℃, 15000rpmで1分間)して再度上清を捨てた。冷却したアセトン(450μL)を加え、遠心後(4℃, 15000rpmで5分間)上清を捨て、さらに遠心(4℃, 15000rpmで1分間)して再度上清を捨てた。これをもう一度繰り返した。遠心エバポレーターで10分間乾燥した後、MilliQ(89μL), 10X DNase buffer(10μL), DNase I(1μL, #2270A, Takara)を加えて、37℃で30分間反応させた。1M硫酸(25μL)を加えて氷上で1時間静置した後、遠心(4℃, 15000rpmで5分間)して上清を回収した。再び遠心(4℃, 15000rpmで1分間)して上清を回収し、先の上清と合わせた。そこに冷却したアセトン(500μL)を加えてよく撹拌したのち、-30℃で一晩静置した。遠心後(4℃, 15000rpmで5分間)上清を捨て、さらに遠心(4℃, 15000rpmで1分間)して再度上清を捨てた。遠心エバポレーターで10分間乾燥した後、重炭酸アンモニウム水溶液(0.1M, 20μL)を加え、そこに直前に調製したプロピオン酸無水物とメタノールの混合物(1:3, 20μL)を加え、さらにアンモニア水(15μL)を加えて室温で30分間静置した。遠心エバポレーターで75分間乾燥した後、消化酵素で処理した。
AB Sciex Triple TOF 4600
Eksigent ekspertTM MicroLC 200
カラム: 3C18-CL-120(0.3mm ID x 150mm)
直線勾配 2%-35%アセトニトリル/0.1%ギ酸, 25分, 5μL/分
サンプル量:5μL
ESI-Q-TOF MS, positive-ion mode.
(3)収率の決定方法
収率の決定方法の概要を図4に示した。
プリカーサーイオンを指定し、それぞれのプリカーサーイオンに対し最も強度の高い2つから4つのMS/MSフラグメントイオンを選択した。指定したプリカーサーイオンおよびMS/MSフラグメントイオンでイオン抽出(±0.5Da)し、以下の式に従って収率を算出した。それを選択したMS/MSフラグメントの全てについて行い、それらの平均を最終収率として決定した。
(ここで、Aはアセチル化ペプチドのピークエリアを示し、Pはプロピオニル化ペプチドのピークエリアを示す)
ひとつのペプチド断片に3つのリジンを含む場合、中央のリジンの収率は
[端2つのリジンどちらかにアセチル化が進行した収率-最端のリジンの収率]
として計算した。
Information Dependent Acquisition Modeで測定を行うことで得られたクロマトグラムに対し、プリカーサーイオンを指定してイオン抽出(±0.5Da)したピークエリアから以下の式に従って収率を決定した。
(ここで、Aはアセチル化ペプチドのピークエリアを示し、Pはプロピオニル化ペプチドのピークエリアを示す)
Method C
Information Dependent Acquisition Modeで測定を行うことで得られたクロマトグラムにおいて、プロピオニル化ペプチド断片は観測されるが、アセチル化ペプチド断片は観測されなかった。このため、サンプルを希釈することでプロピオニル化ペプチド断片の検出限界を算出し、アセチル化収率はその検出限界以下であるとして見積もった。
化合物3(2.30g, 16.0mmol)を12Mメチルアミン水溶液(26.7ml)に溶解し、封管中、120度で18時間加熱した。溶媒を減圧留去したのち、残渣を塩化メチレン/メタノール溶液(4/1)に溶解し、炭酸カリウム(5g)を加えて4時間撹拌した。炭酸カリウムを濾過により除き、ろ液を濃縮することで化合物4(2.01g, 14.5mmol, 88%収率)を得た。
Methyl 3-((2-(hydroxymethyl)pyridin-4-yl)(methyl)amino)propanoate (5):
化合物4(1.38g, 10.0mmol)をアクリル酸メチル(22.5ml)に溶解し、80度で18時間加熱した。溶媒を減圧留去したのち、シリカゲルカラムクロマトグラフィー(酢酸エチル/メタノール=1/0〜0/1)で精製し、化合物5(1.39g, 6.20mmol, 62%収率)を得た。
Methyl 3-((2-(chloromethyl)pyridin-4-yl)(methyl)amino)propanoate (6):
化合物5(640mg, 2.85mmol)を塩化メチレン(14.0ml)に溶解し、塩化チオニル(0.311ml, 4.28mmol)をゆっくりと滴下した。室温で10時間撹拌した後、炭酸カリウム水溶液を注意深く加え、水層を塩化メチレンで抽出、有機層を濃縮し、残渣をシリカゲルクロマトグラフィー(酢酸エチル/メタノール=1/0〜0/1)で精製し、化合物6(529mg, 2.18mmol, 76%収率)を得た。
Methyl 3-(methyl(2-((tritylthio)methyl)pyridin-4-yl)amino)propanoate (7):
化合物6(922mg, 3.80mmol)を塩化メチレン(19.0ml)に溶解し、TrtSH(1.58g, 5.72mmol)およびDBU(0.852ml, 5.70mmol)を加え、室温で10時間撹拌した。炭酸カリウム水溶液を注意深く加え、水層を塩化メチレンで抽出、有機層を濃縮し、残渣をシリカゲルクロマトグラフィー(酢酸エチル/ヘキサン=1/1〜酢酸エチル/メタノール=20/1)で精製し、化合物7(1.53g, 3.17mmol, 83%収率)を得た。
3-(Methyl(2-((tritylthio)methyl)pyridin-4-yl)amino)propanoic acid (8):
化合物7(1.45g, 3.00mmol)をメタノール(15ml)に溶解し、2M水酸化ナトリウム水溶液(7.50ml, 15.0mmol)を加えて、室温で3時間撹拌した。1M塩酸で中和した後、溶媒を留去し、シリカゲルクロマトグラフィー(酢酸エチル/メタノール=4/1〜1/2)で精製し、化合物8(1.39g, 2.97mmol, 99%収率)を得た。
HATU, iPr2NEtを縮合剤、1-Methyl-1H-imidazole-2-carboxylic acid, 4-Amino-1-methyl-1H-pyrrole-2-carboxylic acid, 4-[(9-Fluorenylmethoxycarbonyl)amino]butanoic acidをビルディングブロックとして用いて、通常のFmoc-ペプチド固相合成法に従い2-Chlorotrityl chloride resin(100mg, 1.3mmol/g, 0.133mmol)上にPIP鎖を伸長した。TFA/TIPS/H2O(95/2.5/2.5)で樹脂からの切り出しを行い、HPLC(10% アセトニトリル/0.1% TFA水溶液→直線勾配10-100% 40分, flow: 10ml/分, 230nm, YMC-Pack ODS-AM: 20mm ID x 250mm)で精製しPIP-amine(9, 31.4mg, 0.0204mmol, 15%収率)を得た。
PIP-DSTrt (10):
PIP-amine(15.4mg, 0.0100mmol)をDMF(0.18ml)に溶解し、8(23.4mg, 0.0500mmol), HATU(19.0mg, 0.0500mmol), iPr2NEt(0.0200ml, 0.115mmol)を加えて室温で9時間撹拌した。反応液を濃縮後、HPLC(10% アセトニトリル/0.1% TFA水溶液→直線勾配10-100% 40分, flow: 10ml/分, 230nm, YMC-Pack ODS-AM: 20mm ID x 250mm)で精製しPIP-DSTrt(10, 11.7mg, 0.00587mmol, 59%収率)を得た。
PIP-DSH (1):
PIP-DSTrt(11.7mg, 0.00580mmol)にTIPS(20μl), MilliQ water(20μl)およびTFA(0.160ml)を加え、室温で30分撹拌した。反応液を濃縮後、HPLC(10% アセトニトリル/0.1% TFA水溶液→直線勾配 10-100% 40min, flow: 10ml/min, 230nm, YMC-Pack ODS-AM: 20mm ID x 250mm)で精製しPIP-DSH(1, 6.85mg, 0.00393mmol, 67%収率)を得た。
DIC, HOBtを縮合剤として用いて、通常のFmoc-ペプチド固相合成法に従いRink-Amide-AM resin(75.3mg, 0.73mmol/g, 0.055mmol)上にH-GMRLRSGRSTG-を合成した。HATU, iPr2NEtを用いてN末端に化合物8を縮合させることで、樹脂状に保護LANA-DSHを合成した。TFA/TIPS/H2O(95/2.5/2.5)で脱保護および切り出しを行い、HPLC(直線勾配; 0-75% アセトニトリル/0.1% TFA水溶液, flow: 7ml/分, 30分, 254nm, YMC-Pack ODS-AM: 20mm ID x 250mm)で精製しLANA-DSH(2, 10.5mg, 0.00570mmol, 10%収率)を得た。
[B.結果]
まず用いるアセチル化剤の反応性について検討した(図1)。数種類のアセチル化剤(図1A)について、生細胞より取得した細胞質画分と混合し、アセチル化リジン抗体を用いたウエスタンブロットにより検出した。その結果、EG5-PTAやNMDは多くのタンパク質を非選択的にアセチル化した一方、生体が用いているアセチル基供与体であるアセチルCoAやその類縁体であるTEG-Acはそのような非選択的なアセチル化を引き起こさないことが判明した(図1B)。
Claims (2)
- 下記構造を有する化合物。
- 請求項1に記載の化合物とアシルCoA若しくはその誘導体との組み合わせを含む、タンパク質をアシル化するための薬剤であって、アシルCoAの誘導体が下記構造を有する化合物である薬剤。
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