JP4190753B2 - Search method for plasminogen fragment having angiogenesis inhibitory activity and compound leading to its production - Google Patents

Description

【００１５】
実施例２
化合物４、８、２２および２４の作用によるプラスミノゲンの断片化（図２）。
【００１６】
図２ＡおよびＢに結果を示す対照の実験ではプラスミノゲン（１００ｎＭ）、ｕＰＡ（５０ＩＵ／ｍｌ）を、１００ｍＭ ＮａＣｌおよび０．０１％ Ｔｗｅｅｎ８０を含む５０μｌのトリス塩酸緩衝液（ｐＨ ７．４）中３７°Ｃでインキュベートし、反応０から６０分後に反応液の一部（４５μｌ）を取った。これに５μｌの１ｍＭ Ｖａｌ−Ｌｅｕ−Ｌｙｓ−ｐ−ニトロアニリドを加え、３７°Ｃでインキュベートし随時（１から３分ごとに）４０５ｎｍの吸収をマイクロプレートリーダーで測定した。得られた直線の傾きからプラスミン濃度を算出した（図２Ａ）。また、同時に反応液の一部（２１０μｌ）を７０μｌの４０％トリクロロ酢酸と混合して蛋白質を遠心により回収した。これをアセトンで洗浄後１１μｌのＳＤＳサンプルバッファー（２％ ＳＤＳ、５％ ２−メルカプトエタノール、１０％ショ糖、０．０２％ブロモフェノールブルー（ｂｒｏｍｏｐｈｅｎｏｌ ｂｌｕｅ）を含む６２．５ ｍＭトリス塩酸，ｐＨ６．８）と混合し、その後１０％のポリアクリルアミドゲルを用いたＳＤＳゲル電気泳動によりプラスミノゲンの断片化を観察した（図２Ｂ）。最初の反応液に、化合物４（２５０μＭ）、化合物８（６０μＭ）、化合物２２（３０μＭ）、および化合物２４（０．３μＭ）を用いて得られた結果を、図２Ａ、Ｃ、Ｄ、ＥおよびＦにそれぞれ示す。
【００１７】
図２Ａに示すように、プラスミノゲンの活性化により生ずるプラスミン濃度は経時的に上昇した。これと一致して、プラスミンのＡ鎖およびＢ鎖の増加がＳＤＳゲル電気泳動で確認された（図２Ｂ）。これに対してＳＭＴＰ−６存在下では、プラスミン活性は一過性に上昇したが、その後減少に転じた（図２Ｃ）。活性の減少に伴い、プラスミンＢ鎖の量の減少がＳＤＳゲル電気泳動で示されたが、Ａ鎖の量は経時的に増加した（図２Ｄ）。この結果は、化合物４の存在下ではプラスミンＡ鎖は比較的安定なものの、Ｂ鎖は断片化することを示している。同様に、化合物８、化合物２２、および化合物２４の存在下でもプラスミン濃度の増加は反応１０から３０分後以降見られなくなり（図２Ａ）、これと一致してプラスミンＡ鎖に対するＢ鎖の割合が減少し（図２Ｄ、２Ｅ、２Ｆ）、Ｂ鎖の断片化が顕著に誘導されることが示された。
【００１８】
実施例３
プラスミノゲン断片の構造解析（図３、図４、表２、表３）。
【００１９】
実験例２に記載の方法で得られたプラスミノゲン断片を、還元条件下で２０％ポリアクリルアミドゲルを用いたＳＤＳゲル電気泳動（図３）で分画し、それをＰＶＤＦ（ｐｏｌｙｖｉｎｉｌｉｄｅｎｅ ｄｉｆｌｕｏｒｉｄｅ）膜にブロット後クマシーブリリアントブルー（Ｃｏｏｍａｓｓｉｅ Ｂｒｉｌｌｉａｎｔ Ｂｌｕｅ）Ｒ−２５０で染色した。プラスミノゲン断片のバンド（図３の１から５の矢印）を切り出し、プロテインシークエンサーでＮ末端アミノ酸配列を分析し、表２の結果を得た。また、実験例２に記載の方法で得られたプラスミノゲン断片を凍結乾燥後、還元カルボキシメチル化し、これをＺｉｐＴｉｐ（Ｍｉｌｌｉｐｏｒｅ）を用いて脱塩後、質量分析（ｍａｔｒｉｘ−ａｓｓｏｃｉａｔｅｄｌａｓｅｒ ｄｅｓｏｒｐｔｉｏｎ ｉｏｎｉｚａｔｉｏｎ ｔｉｍｅ ｏｆｆｌｉｇｈｔ ｍａｓｓ（ＭＡＬＤＩ−ＴＯＦ−ＭＳ）ｓｐｅｃｔｒｏｍｅｔｒｙ）により分析した。得られた結果を表３に示す。
【００２０】
【表２】

【００２１】
【表３】

【００２２】
得られた情報を総合して、プラスミノゲン断片の構造を図４に示すように決定した。この結果は、活性化合物の存在下に生成するプラスミノゲン断片は、部分的に断片化したプラスミンＡ鎖とジスルフィド結合で連結したＢ鎖の種々の断片からなる不均一な分子であること示している。これまでにこのような物質は報告されていない。
【００２３】
実施例４
プラスミノゲン断片の精製（図５）。
【００２４】
実験例２に記載の方法で得られたプラスミノゲン断片を含む反応液を水に対して４°Ｃで一晩透析し、これを凍結乾燥した。得られた乾燥物を０．１％トリフルオロ酢酸溶液に溶解後、μＢＯＮＤＡＳＰＨＥＲＥ ５μ Ｃ８−３００Åカラム（１９ ｘ １５０ ｍｍ； Ｗａｔｅｒｓ）を用いた高速液体クロマトグラフィーで分画した。この際、展開溶媒として２−プロパノールと０．１％トリフルオロ酢酸溶液の混合溶媒を用い、流速５ｍｌ／分で２−プロパノール濃度を０％で２０分送液した後、毎分０．８％の割合で増加させた。検出は２１０ｎｍの吸収および２８７ｎｍで励起した３４８ｎｍの蛍光の測定により行なった。溶出時間約６０から６２．５分のピークを分取し、これを凍結乾燥した。これを、０．４５μＭの孔経のフィルターで濾過滅菌後、７．５％ポリアクリルアミドゲルを用いたＳＤＳゲル電気泳動により非還元条件で分析した（図５）。
【００２５】
電気泳動の結果、精製したプラスミノゲン断片の分子量は６６から７２ｋＤａにまたがることが判明した（図５）。
【００２６】
実施例５
プラスミノゲン断片による血管内皮細胞の増殖、遊走、管腔形成の阻害（図６）。
【００２７】
以下の（１）の実験では、実施例４の方法で精製したプラスミノゲン断片を用いた。また（２）、（３）および（４）の実験に用いたプラスミノゲン断片は次の方法で調製された。プラスミノゲン（１００ｎＭ）を化合物４（２５０μＭ）あるいは化合物８（６０μＭ）とともに、ｕＰＡ（５０ＩＵ／ｍｌ）、１００ｍＭ ＮａＣｌおよび０．０１％ Ｔｗｅｅｎ８０を含むＴｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．４）中で３７°Ｃで１時間インキュベート後、反応液に１ｍＭとなるようにフェニルメタンスルホニルふるおらいど（ｐｈｅｎｙｌｍｅｔａｎｅｓｕｌｆｏｎｙｌｆｌｕｏｒｉｄｅ）を加え、精製水に対して４°Ｃで一晩透析した。得られた透析液を凍結乾燥後、０．４５μＭの孔経のフィルターで濾過滅菌した。
【００２８】
（１）ウシ毛細血管内皮細胞（ＢＣＥＣ）の増殖阻害。ＢＣＥＣ（１２，５００細胞）を２４穴組織培養プレートに０．５ｍｌの１０％ウシ胎児血清を含むダルベッコの改変イーグル培地（Ｄｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ ｍｅｄｉｕｍ； ＤＭＥＭ）を用いて播き込んだ。この細胞を２４時間培養後、培地を取り除き、実施例４の方法で精製したプラスミノゲン断片（０．４から４０μｇ／ｍｌ）と０．２５ｍｌの５％ウシ胎児血清を含むＤＭＥＭを加え、２０分間インキュベートした。その後、さらに３ｎｇ／ｍｌの塩基性繊維芽細胞増殖因子（ｂＦＧＦ）と５％ウシ胎児血清を含むＤＭＥＭを０．２５ｍｌ加え、３日間培養を続けた。その後、培地を取り除き、１００μｌの０．０５％トリプシンを含むリン酸緩衝生理食塩水を加え、３７°Ｃで５分間放置後、細胞を懸濁して血球計算版で細胞数を測定した。
【００２９】
図６Ａに示すように、実施例４の方法で精製したプラスミノゲン断片は、培養ＢＣＥＣの増殖を０．２−２０μｇ／ｍｌで１７−５３％阻害した。
【００３０】
（２）ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）の増殖阻害。ＨＵＶＥＣは内皮細胞増殖培地（ＥＧＭ；２％ウシ胎児血清、２０ｎｇ／ｍｌ上皮増殖因子（ＥＧＦ）、１μｇ／ｍｌハイドロコルチゾン（ｈｙｄｒｏｃｏｒｔｉｓｏｎｅ）、１２μｇ／ｍｌウシ脳抽出液（ｂｏｖｉｎｅ ｂｒａｉｎ ｅｘｔｒａｃｔ）を含む内皮細胞基礎培地（ｅｎｄｏｔｈｅｌｉａｌ ｃｅｌｌ ｂａｓａｌ ｍｅｄｉｕｍ）（Ｃｌｏｎｅｔｉｃｓ，Ｓａｎ Ｄｉｅｇｏ， ＣＡ，ＵＳＡ）を用いて培養した。トリプシンにより剥離したＨＵＶＥＣ（２，５００細胞）を９６穴組織培養プレートに０．１ｍｌのＥＧＦを含まないＥＧＭを用いて播き込んだ。この細胞を２４時間培養後、培地を取り除き、プラスミノゲン断片（０．６から６μｇ／ｍｌ）あるいはアンジオスタチン（Ｋ１−Ｋ３）（２０μｇ／ｍｌ）と０．１ｍｌのＥＧＦ不含ＥＧＭを加え、２０分間インキュベートした。その後、さらに２０ｎｇ／ｍｌ上皮増殖因子を含むＥＧＭを０．１ｍｌ加え、３日間培養を続けた。その後、培地を取り除き、０．１ｍｌのＥＧＭと１０μｌ ＷＳＴ−１試薬（ＭＴＴ法に基づく細胞増殖測定キット）（同人化学）加え、４時間３７°Ｃでインキュベートした。その後、４０５ｎｍの吸光度を測定し、細胞数の指標とした。
【００３１】
図６Ｂに示すように、化合物４および８の作用で生ずるプラスミノゲン断片は、培養ＨＵＶＥＣの増殖を０．３−３μｇ／ｍｌで６８−８６％阻害した。比較のために用いたアンジオスタチン（Ｋ１−Ｋ３）は、１０μｇ／ｍｌで７４％の増殖阻害を示した。
【００３２】
また、この増殖阻害作用の特異性を確認するため、血管内皮細胞以外の細胞の増殖に対するプラスミノゲン断片およびアンジオスタチン（Ｋ１−Ｋ３）の影響を調べた。用いた細胞は、ＨＴ１０８０ヒトファイブロサルコーマ（ｆｉｂｒｏｓａｒｃｏｍａ）、ＣＨＯ−Ｋ１細胞および初代ラット皮膚繊維芽細胞で、ＨＴ１０８０細胞およびラット繊維芽細胞は１０％ウシ胎児血清を含むＤＭＥＭ培地を、ＣＨＯ−Ｋ１細胞は１０％ウシ胎児血清を含むＲＰＭＩ１６４０培地を用い、血管内皮細胞を用いた実験と同様の方法で細胞増殖活性を測定した。その結果、図６Ｃに示すように、化合物４および８の作用で生ずるプラスミノゲン断片はアンジオスタチン（Ｋ１−Ｋ３）と同様に、１０μｇ／ｍｌでこれらの細胞の増殖に影響しないことが示された。
【００３３】
（３）ＨＵＶＥＣの遊走阻害。ＨＵＶＥＣを血清およびＥＧＦを含まず、０．１％ウシ血清アルブミンを含むＥＧＭで１晩培養後、２０μｇ／ｍｌのプラスミノゲン断片あるいは２０μｇ／ｍｌのアンジオスタチン（Ｋ１−Ｋ３）を添加した同培地に懸濁し、２０分間インキュベートした。１５μｇフィブロネクチンでコートした８μｍの孔経のポリカーボネート膜インサートを、０．１％ウシ血清アルブミンと１０ｎｇ／ｍｌのｂＦＧＦを含む０．５ｍｌのＥＧＭの入った２４穴組織培養プレートに差し込み、ポリカーボネート膜上に上記の細胞懸濁液０．５ｍｌ（１１０，０００細胞を含む）を播き込んだ。これを５時間培養し、ポリカーボネート膜インサートを取り出した。膜上面の細胞を綿棒で取り除いた後に、膜を通して移動した細胞をギムザで染色し、顕微鏡観察（１００倍）した。
【００３４】
図６Ｄに示すように、化合物４および８の作用で生ずるプラスミノゲン断片は、培養ＨＵＶＥＣの遊走を１０μｇ／ｍｌで２３−３１％阻害した。なお、比較のために用いたアンジオスタチン（Ｋ１−Ｋ３）は３７％の阻害を示した。
【００３５】
（４）ＨＵＶＥＣの管腔形成阻害。２４穴組織培養プレートに、２．６％ウシ胎児血清と０．２４％タイプＩコラーゲンを含む０．２ｍｌ ＭＣＤＢ１３１培地（Ｃｌｏｎｅｔｉｃｓ）入れてゲルを形成させ、これにＨＵＶＥＣ（４０，０００細胞）を０．１ｍｌの２％ウシ胎児血清を含むＭＣＤＢ１３１培地を用いて播き込んだ。この細胞を２時間培養後、培地を取り除き、１０μｇ／ｍｌプラスミノゲン断片あるいは１０μｇ／ｍｌアンジオスタチン（Ｋ１−Ｋ３）、２．６％ウシ胎児血清および０．２４％タイプＩコラーゲンを含む０．２ｍｌ ＭＣＤＢ１３１培地を重層した。３０分後、さらに１０μｇ／ｍｌプラスミノゲン断片と２％ウシ胎児血清を含む０．２ｍｌ ＭＣＤＢ１３１培地を加え２４時間培養した。その後、倒立顕微鏡（４０倍）で細胞を観察した。
【００３６】
図６Ｅに示すように、化合物４および８の作用で生ずるプラスミノゲン断片は、ＨＵＶＥＣの管腔形成を１０μｇ／ｍｌで強く阻害した。比較のために用いたアンジオスタチン（Ｋ１−Ｋ３）にも同様の阻害作用がみられた。
【００３７】
【発明の効果】
本発明により、プラスミノゲンのアンジオスタチン様活性をもつ断片への変換を促進する化合物の有効かつ効率的な探索が可能となった。また、この方法により同定された化合物の作用により生ずるプラスミノゲン断片は新規な血管新生阻害物質であり、これを提供することを可能とした。
【図面の簡単な説明】
【図１】活性測定結果の一例。
【図２】活性化合物の作用によるプラスミノゲン活性化の促進とその結果生ずるプラスミンの断片化。Ａには化合物４（ＳＭＴＰ−６）、８（ｐｌａｃｔｉｎ Ｄ）、２２（ｔｈｉｏｐｌａｂｉｎ Ｂ）および２４（ｃｏｍｐｌｅｓｔａｔｉｎ）の作用によるプラスミノゲン活性化の経時変化を、ＢからＦにはそれぞれ、対照（化合物無添加）および化合物４、８、２２、２４の存在下での反応のＳＤＳゲル電気泳動の結果を示す。図中の矢印ＡはプラスミンのＡ鎖、矢印ＢはＢ鎖の位置を示す。
【図３】活性化合物の作用により生ずるプラスミノゲン断片の解析。化合物４（ＳＭＴＰ−６）、８（ｐｌａｃｔｉｎ Ｄ）、２２（ｔｈｉｏｐｌａｂｉｎＢ）および２４（ｃｏｍｐｌｅｓｔａｔｉｎ）の作用により生ずるプラスミノゲン断片を、還元条件下で２０％ポリアクリルアミドゲルを用いたＳＤＳゲル電気泳動で分画した。図中の矢印ＢはプラスミンのＢ鎖を示す。
【図４】活性化合物の作用により生ずるプラスミノゲン断片の構造の数例太い実線はペプチド鎖を示し、細い実線はシステイン残基間のジスルフィド結合を表す。各アミノ酸は１文字表記で表されている。
【図５】活性化合物の作用により生ずるプラスミノゲン断片の精製。化合物４（ＳＭＴＰ−６）、８（ｐｌａｃｔｉｎ Ｄ）、２２（ｔｈｉｏｐｌａｂｉｎＢ）および２４（ｃｏｍｐｌｅｓｔａｔｉｎ）の作用により生ずるプラスミノゲン断片をμＢＯＮＤＡＳＰＨＥＲＥ ５μ Ｃ８−３００Åカラムを用いた高速液体クロマトグラフィーで精製後、非還元条件下で７．５％ポリアクリルアミドゲルを用いたＳＤＳゲル電気泳動で分析した。Ｍは分子量マーカー、Ｐｌｇはプラスミノゲンを示す。レーン１は化合物４存在下に生ずるプラスミノゲン断片（精製前）、レーン２はその精製後のサンプル、レーン３は化合物２４存在下に生ずるプラスミノゲン断片（精製前）、レーン４はその精製後のサンプル、レーン５は化合物２２存在下に生ずるプラスミノゲン断片（精製前）、レーン６はその精製後のサンプル、レーン７は化合物８存在下に生ずるプラスミノゲン断片（精製前）、レーン８はその精製後のサンプル。
【図６】活性化合物の作用により生ずるプラスミノゲン断片による血管内皮細胞の増殖、遊走および管腔形成の阻害。ＡはＢＣＥＣの、ＢはＨＵＶＥＣの、ＣはＨＴ１０８０細胞、ラット繊維芽細胞ならびにＣＨＯ−Ｋ１細胞の増殖に対する影響を示す。ＤはＨＵＶＥＣの遊走、ＥはＨＵＶＥＣの管腔形成に対する影響を示す。ＡＳ（Ｋ１−Ｋ３）はアンジオスタチン（Ｋ１−Ｋ３）を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for searching for a compound that promotes the production of a plasminogen fragment having an angiogenesis inhibitory action, and the use of a compound having this activity.
[0002]
[Prior art]
Angiostatin is a protein having a molecular weight of about 40,000 (a single polypeptide from the first kringle region to the fourth kringle region) of plasminogen (an inactive precursor of the fibrinolytic enzyme plasmin) that undergoes limited degradation. Inhibits angiogenesis and suppresses cancer growth and metastasis. Angiostatin has shown dramatic effects in animal studies and is currently in clinical trials in the United States. Similar activities have also been reported for plasminogen partial degradation products such as kringle 1-3, kringle 1-5, and kringle 5.
[0003]
Cancer treatment with angiostatin is performed by intravenous administration of purified angiostatin (recombinant protein using E. coli or highly purified elastase digestion fragment of plasma plasminogen). Both purification methods are expensive, and angiostatin production by elastase digestion cannot produce angiostatin selectively due to the low substrate specificity of elastase. The activity of the produced angiostatin does not appear with good reproducibility. Angiostatin is a protein and must be administered directly into the blood. Furthermore, systemic angiostatin administration is difficult to inhibit angiogenesis specifically in cancer tissues. In contrast, compounds that promote the production of angiostatin or plasminogen fragments with similar activity in vivo, mainly occurring in cancer tissues, are thought to exert their actions depending on the regulatory ability of the body. It is expected to exhibit more effective angiogenesis inhibitory activity (anticancer effect) according to various pathological conditions such as cancer progression.
[0004]
Examples of compounds having such activity have not been known so far. In order to search for and identify this, for example, a reaction in which angiostatin is generated from plasminogen (see the section on means for solving the following problems) is reconstructed in vitro and generated. Plasminogen fragments must be analyzed by methods such as SDS polyacrylamide gel electrophoresis, which requires a great deal of labor, time and expense.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide an effective and simple method for searching for a substance that promotes the conversion of plasminogen to a fragment having angiostatin-like activity, and to use a compound identified by this method.
[0006]
[Means for Solving the Problems]
Plasminogen is a single-chain glycoprotein of about 92 kDa, and is composed of an N-terminal peptide (NTP), five kringle regions (K1, K2, K3, K4, K5) and a serine protease region in this order from the amino terminal side. The active enzyme plasmin is obtained by hydrolysis between Arg561 and Val562 by plasminogen activator, and expresses many physiological functions such as thrombolysis, tissue reconstruction, wound healing, and ovulation. On the other hand, a plasminogen fragment having a kringle region (angiostatin consisting of K1-K5, K1-K3, K1-K4.5, K5, etc.) suppresses the proliferation, migration, and lumen formation of vascular endothelial cells, thereby causing angiogenesis. Inhibit. Due to this action, angiostatin is currently in clinical development as an anticancer agent. Angiostatin production from plasminogen proceeds by the following pathway. Plasminogen is activated to plasmin by urokinase-type plasminogen activator (uPA), which is abundant in tumor tissue, a part of which is reduced by the action of phosphoglycerate kinase in the tumor tissue and the disulfide bond in K5 is cleaved. This molecule is subjected to limited degradation by plasmin or the like to become K1-K4.5, which is further subjected to limited degradation by matrix metalloproteinase (MMP) in the tumor tissue to become K1-K4 (angiostatin). Since plasminogen in the circulating blood has an intramolecular bond between NTP and K5 and takes a tight conformation, it exhibits resistance to activation by plasminogen activator. The inventor can promote (1) conversion of plasminogen to plasmin and (2) fragmentation of plasminogen by a protease such as plasmin by relaxing the conformation of plasminogen, and has the same activity as angiostatin or angiostatin We hypothesized that it would be possible to generate plasminogen fragments with That is, it was considered that a compound that promotes the production of a plasminogen fragment having angiostatin-like activity could be discovered using the promotion of plasminogen activation as an index. In order to confirm this, a compound that promotes this activity was searched from a culture extract of about 5000 strains of microorganisms using an in vitro reaction system for measuring activation of plasminogen. As a result, staprabin (compound 1 according to claim 2) and SMTP-4 to 8 (compounds 2 to 6 according to claim 2) produced by the Stachybotrys microspora IFO30018 strain, and compounds that are incompletely spore-free Plactins B and D (compounds 7 and 8 according to claim 2) produced by the fungus F164 strain, thioplabins A, B and C produced by the Streptomyces sp. Strain R1401 Compound 21 to 23 according to claim 2), complexstatin and chloropeptin (chlorop) produced by Streptomyces sp. Strain A1631 PTIN) was identified I (compound 24 and 25 according to claim 2) as active substance. Further, as a result of examining the activity of the chemically synthesized plactin derivative, it was clarified that the compounds 9 to 20 according to claim 2 have the same activity.
[0007]
These compounds transiently promoted plasmin production transiently when plasminogen was activated with uPA, but resulted in subsequent fragmentation of plasmin. The generated plasminogen fragment spanned a molecular weight of 50 kDa to 72 kDa, contained K1-K5, and was a novel molecule in which part of the protease region was linked by a disulfide bond. It was revealed that plasminogen fragments obtained by the action of various compounds inhibit proliferation, migration, and lumen formation of cultured vascular endothelial cells in the same manner as angiostatin and are useful as angiogenesis inhibitors.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in more detail.
[0009]
A. The first of the present invention is the development of a method for efficiently searching for compounds that promote the conversion of plasminogen into a fragment having angiostatin-like activity. As described in the item of means for solving the above-mentioned problems, an important point of the present invention is to search for this using the promotion of activation of plasminogen as an index. As a result, automatic measurement using an assay plate having 96 holes or more (384 holes, 1536 holes, etc.) is possible, resulting in a dramatic increase in search efficiency.
[0010]
B. The second of the present invention is a plasminogen fragment produced by the action of a compound identified by the above method (compounds 1 to 25 according to claim 2). This is a novel molecule spanning a molecular weight of 50 kDa to 72 kDa, containing K1-K5, and further linking a part of the protease region with a disulfide bond, and is capable of controlling proliferation, migration, and luminal formation of cultured vascular endothelial cells with angiostatin. Similarly inhibited.
【Example】
Examples of the present invention will be specifically described below.
[0011]
Example 1
An efficient search method for a compound that promotes the conversion of plasminogen into a fragment having angiostatin-like activity.
[0012]
The method for measuring activity using a 96-well assay plate is shown below. 96-well assay with 50 μl Tris-HCl buffer (pH 7.4) containing 100 nM plasminogen, 50 IU / ml uPA, 100 μM Val-Leu-Lys-p-nitroanilide (plasmin substrate), 100 mM NaCl and 0.01% Tween 80 The plate was incubated at 37 ° C. and the absorbance at 405 nm was measured with a microplate reader as needed (every 1 to 3 minutes). The initial reaction rate (plasmin production rate) can be determined by taking the absorbance obtained on the vertical axis and the square of time on the horizontal axis. An aliquot of the microorganism culture extract was dried under reduced pressure and then added to the reaction solution. In addition, the numerical value of an Example shows an example, The density | concentration of each component in this measuring system, the component and pH of a buffer solution, the quantity of reaction liquid, reaction temperature, reaction time can be changed suitably, and this invention Is not limited to these values. In addition, N-terminal lysine-type plasminogen (Lys-plasminogen) or plasma (about 1% to 50% in final concentration) is used instead of plasminogen, and other plasminogen activator (such as tissue-type plasminogen activator) is used instead of uPA. Can also be used.
[0013]
As a result of searching for a compound that promotes this activity from the culture extract of about 5000 strains of microorganisms, culture of Streptomyces sp. R1401 strain, Streptomyces sp. A1631, Spore-incomplete fungus F164 strain, Stachybotrys microspora IFO30018 strain Strong activity was observed in the liquid. As a result of purifying and isolating the active substance and determining its structure, the compounds 1 to 8 and 21 to 25 according to claim 2 were identified. Moreover, as a result of investigating the activity of the chemically synthesized plactin derivative, it was shown that the compounds 9 to 20 according to claim 2 have the same activity (Table 1). FIG. 1 shows an example of the results of this activity measurement.
[0014]
[Table 1]

[0015]
Example 2
Fragmentation of plasminogen by the action of compounds 4, 8, 22 and 24 (FIG. 2).
[0016]
In a control experiment, the results of which are shown in FIGS. 2A and B, plasminogen (100 nM), uPA (50 IU / ml), 37 ° in 50 μl Tris-HCl buffer (pH 7.4) containing 100 mM NaCl and 0.01% Tween 80. Incubation was performed at C, and a part of the reaction solution (45 μl) was taken after 60 minutes from the reaction. To this was added 5 μl of 1 mM Val-Leu-Lys-p-nitroanilide, incubated at 37 ° C., and the absorbance at 405 nm was measured with a microplate reader as needed (every 1 to 3 minutes). The plasmin concentration was calculated from the slope of the obtained straight line (FIG. 2A). Simultaneously, a part of the reaction solution (210 μl) was mixed with 70 μl of 40% trichloroacetic acid, and the protein was recovered by centrifugation. This was washed with acetone, and 11 μl of SDS sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% sucrose, 62.5 mM Tris-HCl containing 0.02% bromophenol blue, pH 6. 8), and then fragmentation of plasminogen was observed by SDS gel electrophoresis using 10% polyacrylamide gel (FIG. 2B). The results obtained using Compound 4 (250 μM), Compound 8 (60 μM), Compound 22 (30 μM), and Compound 24 (0.3 μM) in the initial reaction are shown in FIGS. 2A, C, D, E and Each is shown in F.
[0017]
As shown in FIG. 2A, the plasmin concentration produced by plasminogen activation increased with time. Consistent with this, increases in plasmin A and B chains were confirmed by SDS gel electrophoresis (FIG. 2B). In contrast, in the presence of SMTP-6, plasmin activity increased transiently, but then turned to decrease (FIG. 2C). With decreasing activity, SDS gel electrophoresis showed a decrease in the amount of plasmin B chain, but the amount of A chain increased over time (FIG. 2D). This result indicates that in the presence of Compound 4, the plasmin A chain is relatively stable, but the B chain is fragmented. Similarly, in the presence of Compound 8, Compound 22, and Compound 24, the increase in plasmin concentration is not observed after 30 minutes from the reaction 10 (FIG. 2A), and in agreement with this, the ratio of B chain to plasmin A chain is Decreased (FIGS. 2D, 2E, 2F), indicating that B chain fragmentation was significantly induced.
[0018]
Example 3
Structural analysis of plasminogen fragments (FIG. 3, FIG. 4, Table 2, Table 3).
[0019]
The plasminogen fragment obtained by the method described in Experimental Example 2 was fractionated by SDS gel electrophoresis using a 20% polyacrylamide gel under reducing conditions (FIG. 3), and blotted onto a PVDF (polyvinylidene difluoride) membrane. After staining with Coomassie Brilliant Blue R-250. The band of the plasminogen fragment (arrows 1 to 5 in FIG. 3) was cut out and the N-terminal amino acid sequence was analyzed with a protein sequencer, and the results shown in Table 2 were obtained. In addition, the plasminogen fragment obtained by the method described in Experimental Example 2 was lyophilized, reduced carboxymethylated, desalted using ZipTip (Millipore), and then subjected to mass spectrometry (matrix-associated radical desorption time offset mass ( MALDI-TOF-MS) Spectrometry). The obtained results are shown in Table 3.
[0020]
[Table 2]

[0021]
[Table 3]

[0022]
By combining the obtained information, the structure of the plasminogen fragment was determined as shown in FIG. This result indicates that the plasminogen fragment produced in the presence of the active compound is a heterogeneous molecule consisting of various fragments of a partially fragmented plasmin A chain and a B chain linked by disulfide bonds. To date, no such substance has been reported.
[0023]
Example 4
Purification of plasminogen fragment (Figure 5).
[0024]
The reaction solution containing the plasminogen fragment obtained by the method described in Experimental Example 2 was dialyzed against water at 4 ° C. overnight and lyophilized. The obtained dried product was dissolved in a 0.1% trifluoroacetic acid solution, and fractionated by high-performance liquid chromatography using a μBONASPHERE 5μ C8-300 column (19 × 150 mm; Waters). At this time, a mixed solvent of 2-propanol and 0.1% trifluoroacetic acid solution was used as a developing solvent, and the 2-propanol concentration was fed at 0% for 20 minutes at a flow rate of 5 ml / min, and then 0.8% per minute. Increased at a rate of. Detection was performed by measuring absorption at 210 nm and fluorescence at 348 nm excited at 287 nm. A peak with an elution time of about 60 to 62.5 minutes was collected and lyophilized. This was sterilized by filtration with a 0.45 μM pore filter, and analyzed by SDS gel electrophoresis using 7.5% polyacrylamide gel under non-reducing conditions (FIG. 5).
[0025]
As a result of electrophoresis, it was found that the molecular weight of the purified plasminogen fragment ranges from 66 to 72 kDa (FIG. 5).
[0026]
Example 5
Inhibition of vascular endothelial cell proliferation, migration, and lumen formation by plasminogen fragments (FIG. 6).
[0027]
In the following experiment (1), a plasminogen fragment purified by the method of Example 4 was used. The plasminogen fragment used in the experiments (2), (3) and (4) was prepared by the following method. Plasminogen (100 nM) together with Compound 4 (250 μM) or Compound 8 (60 μM) in Tris-HCl buffer (pH 7.4) containing uPA (50 IU / ml), 100 mM NaCl and 0.01% Tween 80 at 37 ° C. After incubating for 1 hour, phenylmethanesulfonyl fluoride was added to the reaction solution to 1 mM and dialyzed against purified water overnight at 4 ° C. The resulting dialysate was freeze-dried and then sterilized by filtration through a 0.45 μM pore filter.
[0028]
(1) Growth inhibition of bovine capillary endothelial cells (BCEC). BCEC (12,500 cells) were seeded in 24-well tissue culture plates using Dulbecco's modified Eagle medium (DMEM) containing 0.5 ml of 10% fetal calf serum. After culturing the cells for 24 hours, the medium was removed, plasminogen fragment (0.4 to 40 μg / ml) purified by the method of Example 4 and 0.25 ml of DMEM containing 5% fetal calf serum were added, and incubated for 20 minutes. did. Thereafter, 0.25 ml of DMEM containing 3 ng / ml basic fibroblast growth factor (bFGF) and 5% fetal calf serum was added, and the culture was continued for 3 days. Thereafter, the medium was removed, 100 μl of phosphate buffered saline containing 0.05% trypsin was added, the mixture was allowed to stand at 37 ° C. for 5 minutes, the cells were suspended, and the number of cells was measured with a hemocytometer.
[0029]
As shown in FIG. 6A, the plasminogen fragment purified by the method of Example 4 inhibited the growth of cultured BCEC by 17-53% at 0.2-20 μg / ml.
[0030]
(2) Inhibition of proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC is an endothelial cell containing endothelial cell growth medium (EGM; 2% fetal bovine serum, 20 ng / ml epidermal growth factor (EGF), 1 μg / ml hydrocortisone, 12 μg / ml bovine brain extract) Cultured with basal medium (endotic cell basal medium) (Clonetics, San Diego, Calif., USA) HUVECs (2,500 cells) exfoliated with trypsin do not contain 0.1 ml EGF in 96-well tissue culture plates After culturing the cells for 24 hours, the medium was removed, and the plasminogen fragment (0.6 to 6 μg / ml) or angiostatin (K1-K3) (20 μg / ml) and 0.1 ml of E F-free EGM was added and incubated for 20 minutes, after which another 0.1 ml of EGM containing 20 ng / ml epidermal growth factor was added and the culture was continued for 3 days, after which the medium was removed and 0.1 ml of EGM and 10 μl were added. WST-1 reagent (a cell proliferation measurement kit based on the MTT method) (doujinshi) was added and incubated for 4 hours at 37 ° C. Thereafter, the absorbance at 405 nm was measured and used as an index of the number of cells.
[0031]
As shown in FIG. 6B, the plasminogen fragment produced by the action of compounds 4 and 8 inhibited the growth of cultured HUVECs by 68-86% at 0.3-3 μg / ml. Angiostatin (K1-K3) used for comparison showed 74% growth inhibition at 10 μg / ml.
[0032]
In addition, in order to confirm the specificity of this growth inhibitory action, the effects of plasminogen fragment and angiostatin (K1-K3) on the growth of cells other than vascular endothelial cells were examined. The cells used were HT1080 human fibrosarcoma, CHO-K1 cells and primary rat skin fibroblasts. HT1080 cells and rat fibroblasts were prepared using DMEM medium containing 10% fetal bovine serum, CHO-K1 cells. Used RPMI1640 medium containing 10% fetal bovine serum, and cell proliferation activity was measured by the same method as the experiment using vascular endothelial cells. As a result, as shown in FIG. 6C, it was shown that the plasminogen fragment generated by the action of compounds 4 and 8 did not affect the proliferation of these cells at 10 μg / ml, as in the case of angiostatin (K1-K3).
[0033]
(3) Inhibition of HUVEC migration. HUVECs were cultured overnight in EGM containing no serum and EGF but 0.1% bovine serum albumin, and then suspended in the same medium supplemented with 20 μg / ml plasminogen fragment or 20 μg / ml angiostatin (K1-K3). Turbid and incubated for 20 minutes. An 8 μm pore polycarbonate membrane insert coated with 15 μg fibronectin was inserted into a 24-well tissue culture plate containing 0.5 ml EGM containing 0.1% bovine serum albumin and 10 ng / ml bFGF and placed on the polycarbonate membrane. 0.5 ml of the above cell suspension (containing 110,000 cells) was seeded. This was cultured for 5 hours, and the polycarbonate membrane insert was taken out. After the cells on the upper surface of the membrane were removed with a cotton swab, the cells that migrated through the membrane were stained with Giemsa and observed with a microscope (100 times).
[0034]
As shown in FIG. 6D, the plasminogen fragment produced by the action of compounds 4 and 8 inhibited the migration of cultured HUVEC by 23-31% at 10 μg / ml. The angiostatin (K1-K3) used for comparison showed 37% inhibition.
[0035]
(4) Inhibition of HUVEC tube formation. In a 24-well tissue culture plate, 0.2 ml MCDB131 medium (Clonetics) containing 2.6% fetal bovine serum and 0.24% type I collagen was added to form a gel, and HUVEC (40,000 cells) was added to this. 1. Seeded with MCDB131 medium containing 1 ml of 2% fetal calf serum. After culturing the cells for 2 hours, the medium was removed and 0.2 μl MCDB131 containing 10 μg / ml plasminogen fragment or 10 μg / ml angiostatin (K1-K3), 2.6% fetal bovine serum and 0.24% type I collagen. Medium was overlaid. After 30 minutes, 0.2 ml MCDB131 medium containing 10 μg / ml plasminogen fragment and 2% fetal bovine serum was further added and cultured for 24 hours. Thereafter, the cells were observed with an inverted microscope (40 ×).
[0036]
As shown in FIG. 6E, plasminogen fragments generated by the action of compounds 4 and 8 strongly inhibited HUVEC tube formation at 10 μg / ml. A similar inhibitory action was also observed in angiostatin (K1-K3) used for comparison.
[0037]
【The invention's effect】
According to the present invention, an effective and efficient search for a compound that promotes the conversion of plasminogen into a fragment having angiostatin-like activity has become possible. In addition, the plasminogen fragment generated by the action of the compound identified by this method is a novel angiogenesis inhibitor, which can be provided.
[Brief description of the drawings]
FIG. 1 shows an example of activity measurement results.
FIG. 2. Promotion of plasminogen activation by the action of active compounds and consequent fragmentation of plasmin. A shows the time course of plasminogen activation due to the action of compounds 4 (SMTP-6), 8 (practin D), 22 (thioplabine B) and 24 (complestatin), and B to F each show a control (no compound added). ) And the results of SDS gel electrophoresis of the reaction in the presence of compounds 4, 8, 22, 24. The arrow A in the figure indicates the position of the A chain of plasmin, and the arrow B indicates the position of the B chain.
FIG. 3. Analysis of plasminogen fragments generated by the action of active compounds. Fractionation of plasminogen fragments generated by the action of compounds 4 (SMTP-6), 8 (practin D), 22 (thioplanein B) and 24 (completestatin) by SDS gel electrophoresis using 20% polyacrylamide gel under reducing conditions. did. Arrow B in the figure indicates the B chain of plasmin.
4 shows several examples of the structure of a plasminogen fragment generated by the action of an active compound. A thick solid line indicates a peptide chain, and a thin solid line indicates a disulfide bond between cysteine residues. Each amino acid is represented by a single letter.
FIG. 5 Purification of plasminogen fragments generated by the action of active compounds. After purifying the plasminogen fragment produced by the action of compounds 4 (SMTP-6), 8 (practin D), 22 (thioplabin B) and 24 (completestatin) by high-performance liquid chromatography using a μBONDASPHERE 5μ C8-300 column, non-reducing conditions Under analysis by SDS gel electrophoresis using 7.5% polyacrylamide gel. M represents a molecular weight marker, and Plg represents plasminogen. Lane 1 is a plasminogen fragment generated in the presence of compound 4 (before purification), lane 2 is a sample after purification, lane 3 is a plasminogen fragment generated in the presence of compound 24 (before purification), lane 4 is a sample after purification, Lane 5 is a plasminogen fragment produced in the presence of compound 22 (before purification), lane 6 is a sample after purification, lane 7 is a plasminogen fragment produced in the presence of compound 8 (before purification), and lane 8 is a sample after purification.
FIG. 6: Inhibition of vascular endothelial cell proliferation, migration and lumen formation by plasminogen fragments generated by the action of active compounds. A shows the effect of BCEC, B shows the effect of HUVEC, and C shows the effect on the growth of HT1080 cells, rat fibroblasts and CHO-K1 cells. D shows migration of HUVEC, and E shows the effect of HUVEC on tube formation. AS (K1-K3) indicates angiostatin (K1-K3).

Claims (3)

Promoting the conversion of plasminogen into a fragment with angiostatin-like activity, including contacting the test substance with a substrate for plasminogen, plasminogen activator and plasmin and measuring the activation of plasminogen by the plasminogen activator Compound search method. (I) a polypeptide comprising the amino acid at amino acid position V79-R561 of plasminogen, or
A polypeptide in which a polypeptide comprising amino acids at amino acid positions V79-K208 and N209-R561 of plasminogen is linked by a disulfide bond;
( ii A fragment of a polypeptide consisting of the amino acid at amino acid position V562-N791 of plasminogen,
Amino acid positions of plasminogen V562-R580, V562-K607, V562-K615, V562-K698, V606-R677, V616-K645, V616-K651, V616-K698, L652-K698, L652-K708, V662-R677, V662- Polypeptide selected from the group consisting of K698, E699-R789, E699-N791, V709-R719, V709-N791, V720-K750, V720-R776, V720-R789, V720-N791, D751-R789 or Y753-R776
And a polypeptide linked by a disulfide bond. A compound obtainable by the method of claim 1, comprising:
following:

An agent for promoting the conversion of plasminogen into a fragment having angiostatin-like activity, comprising a compound selected from the group consisting of:

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