JP2012206959A - Composition for inhibiting glucosidase activity and method for screening the same - Google Patents
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本発明は、グルコシダーゼ活性阻害用組成物及びそのスクリーニング方法に関する。さらに、抗ＨＩＶ活性を有するグルコシダーゼ活性阻害用組成物及びそのスクリーニング方法に関する。 The present invention relates to a composition for inhibiting glucosidase activity and a screening method thereof. Furthermore, it is related with the composition for glucosidase activity inhibition which has anti-HIV activity, and its screening method.
インフルエンザウイルスやヒト免疫不全ウイルス（Ｈｕｍａｎ Ｉｍｍｕｎｏｄｅｆｉｃｉｅｎｃｙ Ｖｉｒｕｓ，以下、ＨＩＶとする）等のウイルスは、糖鎖の認識によって、宿主とするヒト等に特異的に結合し、感染することが知られている。そこで、ウイルスの宿主への結合を抑制するために、糖鎖を制御し、非感染性のウイルスとする様々な物質のスクリーニングが行われている。
糖鎖の制御はグルコシダーゼ活性を阻害することで行うことができることから、ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）属に属する微生物の培養液から単離されたα−グルコシダーゼ阻害活性を有する化合物（Ａ−７６２０２）や、薬用植物ディクロスタキス・シネレアから単離されるα−グルコシダーゼ阻害剤化合物（−）−メスキトール等が見出され、抗肥満剤、抗糖尿病剤又は抗ＨＩＶ剤として有用であるとされている（特許文献１、２参照）。
Viruses such as influenza virus and human immunodeficiency virus (hereinafter referred to as HIV) are known to specifically bind to and infect humans or the like as hosts by recognition of sugar chains. Therefore, in order to suppress the binding of the virus to the host, screening of various substances that control sugar chains and make non-infectious viruses has been performed.
Since the sugar chain can be controlled by inhibiting the glucosidase activity, a compound (A-76202) having an α-glucosidase inhibitory activity isolated from a culture solution of a microorganism belonging to the genus Rhodococcus, Α-Glucosidase inhibitor compound (−)-mesquitol isolated from plant diclostaxis cinerea has been found and is said to be useful as an anti-obesity agent, anti-diabetic agent or anti-HIV agent (Patent Document 1). 2).
また、ピパタリン、セサミン、ペリトリン等のα−グルコシダーゼ阻害物質を有する製薬調合物によって、糖尿病、癌、Ｂ型／Ｃ型肝炎、ＨＩＶ、ＡＩＤＳ等の患者にα−グルコシダーゼ阻害効果を提供する方法等も開発されている（特許文献３参照）。
しかし、これらの物質は、グルコシダーゼ阻害活性を有することは確認されているものの、投与における人体等への安全性については示されていない。
また、これらの物質を、例えば抗ＨＩＶ剤として用いる場合においても、他の抗ＨＩＶ剤と３〜４剤を併用する多剤併用療法に用いられる可能性が高い。多剤併用療法では、副作用、服用の複雑さ、耐性菌の出現等様々な欠点があり、さらにＨＩＶを完全に排除できないという問題がある。そこで、優れたグルコシダーゼ阻害活性を有し、ヒト等の摂取において安全性が高く、さらに抗ＨＩＶ剤等に用いる場合には単剤で有効な効果を示す物質の提供が望まれている。
In addition, a method of providing an α-glucosidase inhibitory effect to patients with diabetes, cancer, hepatitis B / C, HIV, AIDS, etc. by a pharmaceutical preparation having an α-glucosidase inhibitor such as pipetalin, sesamin, peritrine, etc. It has been developed (see Patent Document 3).
However, although these substances have been confirmed to have glucosidase inhibitory activity, they have not been shown to be safe for the human body or the like upon administration.
Moreover, even when these substances are used, for example, as anti-HIV agents, there is a high possibility that they will be used in multi-drug combination therapy in which other anti-HIV agents and 3-4 agents are used in combination. Multi-drug combination therapy has various drawbacks such as side effects, complexity of taking, emergence of resistant bacteria, and there is a problem that HIV cannot be completely eliminated. Therefore, it is desired to provide a substance that has an excellent glucosidase inhibitory activity, is highly safe when ingested by humans and the like, and exhibits an effective effect when used alone as an anti-HIV agent.
本発明は、グルコシダーゼ活性阻害用組成物及びそのスクリーニング方法の提供を課題とする。さらに、抗ＨＩＶ活性を有するグルコシダーゼ活性阻害用組成物及びそのスクリーニング方法の提供を課題とする。 An object of the present invention is to provide a composition for inhibiting glucosidase activity and a screening method thereof. Furthermore, it is an object to provide a composition for inhibiting glucosidase activity having anti-HIV activity and a screening method thereof.
本発明者らは、上記課題を解決するために鋭意研究を行った結果、α−グルコシダーゼの立体構造を標的として活性部位を抽出し、その部位に適合する物質を既知の化合物データベースからコンピュータを用いてバーチャルスクリーニングすることにより、優れたグルコシダーゼ阻害活性を有し、ヒト等の摂取において安全性が高く、さらに抗ＨＩＶ活性も有する新規の物質が得られることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above-mentioned problems, the present inventors have extracted an active site by targeting the three-dimensional structure of α-glucosidase, and used a computer from a known compound database for a substance that matches that site. By conducting virtual screening, it was found that a novel substance having excellent glucosidase inhibitory activity, high safety in ingestion by humans and the like and also having anti-HIV activity was obtained, and the present invention was completed. .
すなわち、本発明は次の（１）〜（８）のグルコシダーゼ活性阻害用組成物及び該組成物のスクリーニング方法等に関する。
（１）下記の式［化１］で表される構造を母核として有する化合物であるグルコシダーゼ活性阻害用組成物。
［化１］
（２）さらに抗ＨＩＶ活性を有する上記（１）に記載のグルコシダーゼ活性阻害用組成物。
（３）置換基が、１）Ｒ1-6は水素原子、ハロゲン原子、炭素数１〜１０のアルキル基、炭素数１〜１０のアシル基、炭素数１〜１０のシリル基から選ばれるいずれかであり、２）Ｒ7は水素原子、ハロゲン原子、炭素数１〜１０のアルキル基、炭素数１〜１０のアシル基、炭素数１〜１０のシリル基、環式炭化水素誘導体、縮合多環式炭化水素誘導体、環式ヘテロ炭化水素誘導体、縮合ヘテロ多環式炭化水素誘導体から選ばれるいずれかの化合物である上記（１）又は（２）に記載のグルコシダーゼ活性阻害用組成物。
（４）下記の式［化４］〜［化１３］のいずれかで表される化合物である上記（１）〜（３）のいずれかに記載のグルコシダーゼ活性阻害用組成物。
［化４］
［化５］
［化６］
［化７］
［化８］
［化９］
［化１０］
［化１１］
［化１２］
［化１３］
（５）上記（１）〜（４）のいずれかに記載のグルコシダーゼ活性阻害用組成物を有効成分として含む抗ＨＩＶ剤。
（６）次のスクリーニングルールによってｉｎ ｓｉｌｉｃｏバーチャルスクリーニングを行うことによるグルコシダーゼ活性阻害用組成物のスクリーニング方法。
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位のタンパク質表面を、半径１〜２オングストロームの球を用いて仮想的に覆う
２）活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、化合物とタンパク質の疎水性相互作用を生じる可能性空間とする
３）さらに、活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、リガンドとタンパク質の親水性相互作用を生じる可能性空間とする
４）活性部位内に設置した可能性空間に少なくとも１〜５以上の原子が存在し、かつ、活性部位を逸脱しない化合物の検索を行う
（７）さらに、次のスクリーニングルールによってｉｎ ｓｉｌｉｃｏバーチャルスクリーニングを行うことによるグルコシダーゼ阻害活性を有する上記（６）に記載のグルコシダーゼ活性阻害用組成物のスクリーニング方法。
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位空間に対して、１つの化合物当り１０〜１００００のコンフォマーを仮想的に作成し、活性部位空間に化合物を配置する
２）得られたモデルを水素結合、配位結合、イオン的相互作用、化合物自由度、脱溶媒和エネルギー、化合物の埋没度、物理的な接触及び化合物の基質結合部位に対する充填率について、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアのスコアリング関数によって評価し、上位１／３をヒット化合物とする
３）上記２）で次に、得られた化合物の群に対し、水素結合、配位結合、イオン的相互作用を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする
４）さらに、上記３）で得られた化合物の群に対し、水素結合を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする
（８）さらに、グルコシダーゼ阻害活性をバイオアッセイによってスクリーニングする上記（６）又は（７）に記載のグルコシダーゼ活性阻害用組成物のスクリーニング方法。
That is, the present invention relates to the following glucosidase activity inhibiting composition (1) to (8), a screening method for the composition, and the like.
(1) A composition for inhibiting glucosidase activity, which is a compound having a structure represented by the following formula [Chemical Formula 1] as a mother nucleus.
[Chemical 1]
(2) The composition for inhibiting glucosidase activity according to (1), further having anti-HIV activity.
(3) The substituent is 1) R 1-6 is any one selected from a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, an acyl group having 1 to 10 carbon atoms, and a silyl group having 1 to 10 carbon atoms. 2) R 7 is a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, an acyl group having 1 to 10 carbon atoms, a silyl group having 1 to 10 carbon atoms, a cyclic hydrocarbon derivative, a condensed poly The composition for inhibiting glucosidase activity according to the above (1) or (2), which is any compound selected from cyclic hydrocarbon derivatives, cyclic heterohydrocarbon derivatives, and condensed heteropolycyclic hydrocarbon derivatives.
(4) The composition for inhibiting glucosidase activity according to any one of the above (1) to (3), which is a compound represented by any of the following formulas [Chemical Formula 4] to [Chemical Formula 13].
[Chemical formula 4]
[Chemical formula 5]
[Chemical 6]
[Chemical 7]
[Chemical 8]
[Chemical 9]
[Chemical Formula 10]
[Chemical 11]
[Chemical 12]
[Chemical 13]
(5) An anti-HIV agent comprising the composition for inhibiting glucosidase activity according to any one of (1) to (4) as an active ingredient.
(6) A screening method for a composition for inhibiting glucosidase activity by performing in silico virtual screening according to the following screening rules.
1) The protein surface of the active site estimated from the X-ray crystallographic image of glucosidase is virtually covered with a sphere with a radius of 1 to 2 angstroms. 2) A sphere with a radius of 1 to 2 angstroms is placed in the active site. Place 1-10 spheres to generate a hydrophobic interaction between the compound and protein. 3) Furthermore, place 1-10 spheres with a radius of 1-2 angstroms in the active site to make the hydrophilicity of the ligand and protein. 4) Search for compounds that have at least 1 to 5 atoms in the possibility space installed in the active site and do not deviate from the active site (7) The glucosidase according to (6) above, which has a glucosidase inhibitory activity by performing in silico virtual screening according to the following screening rule: A method for screening a composition for inhibiting zeta activity.
1) Virtually create 10-10000 conformers per compound for the active site space estimated for the X-ray crystallographic image of glucosidase, and place the compound in the active site space 2) The Chemical Computation Group Inc. was used for hydrogen bonds, coordination bonds, ionic interactions, compound degrees of freedom, desolvation energy, compound embedment, physical contact, and packing ratio of the compound to the substrate binding site. 3) Using the top 1/3 as a hit compound, 3) Next, in 2) above, hydrogen bonds, coordination bonds, and ionic interactions are obtained. Is evaluated by the same scoring function, and the top 1/3 is regarded as a hit compound. 4) Further, for the group of compounds obtained in the above 3), the hydrogen bond is emphasized. And using the same scoring function to evaluate, and using the top 1/3 as a hit compound (8) Furthermore, screening for glucosidase inhibitory activity by bioassay is for glucosidase activity inhibition according to (6) or (7) above Composition screening method.
本発明のグルコシダーゼ活性阻害用組成物は、優れたグルコシダーゼ阻害活性を有する新規な物質であり、糖鎖を制御できることから、糖鎖生物学における研究用試薬等に用いることができる。また、ヒト等の摂取において安全性が高く、さらに抗ＨＩＶ活性を有することから、抗ＨＩＶ剤の有効成分として用いることができる。 The composition for inhibiting glucosidase activity of the present invention is a novel substance having excellent glucosidase inhibitory activity and can control sugar chains, and therefore can be used as a research reagent in sugar chain biology. Further, since it is highly safe for human consumption and has anti-HIV activity, it can be used as an active ingredient of anti-HIV agents.
本発明の「グルコシダーゼ活性阻害用組成物」とは、下記の式［化１］で表される構造を母核として有するグルコシダーゼ活性阻害用組成物であればいずれのものも含まれる。 The “composition for inhibiting glucosidase activity” of the present invention includes any composition as long as it is a composition for inhibiting glucosidase activity having a structure represented by the following formula [Chemical Formula 1] as a mother nucleus.
［化１］
[Chemical 1]
本発明の「グルコシダーゼ活性阻害用組成物」は抗ＨＩＶ活性を有している物質であることが好ましく、さらに細胞毒性の低い、人体等に安全な物質であることが好ましい。
このような物質として、置換基が、１）Ｒ1-6は水素原子、ハロゲン原子、炭素数１〜１０のアルキル基、炭素数１〜１０のアシル基又は炭素数１〜１０のシリル基から選ばれるいずれかからなる化合物や、２）Ｒ7は水素原子、ハロゲン原子、炭素数１〜１０のアルキル基、炭素数１〜１０のアシル基、炭素数１〜１０のシリル基、環式炭化水素誘導体、縮合多環式炭化水素誘導体、環式ヘテロ炭化水素誘導体又は縮合ヘテロ多環式炭化水素誘導体から選ばれるいずれかからなる化合物が挙げられる。さらに、化学式４〜化学式１３（［化４］〜［化１３］）で表される化合物等が挙げられる。
The “composition for inhibiting glucosidase activity” of the present invention is preferably a substance having anti-HIV activity, and more preferably a substance having low cytotoxicity and safe for the human body.
As such a substance, the substituent is 1) R 1-6 is a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, an acyl group having 1 to 10 carbon atoms or a silyl group having 1 to 10 carbon atoms. 2) R 7 is a hydrogen atom, a halogen atom, an alkyl group having 1 to 10 carbon atoms, an acyl group having 1 to 10 carbon atoms, a silyl group having 1 to 10 carbon atoms, or cyclic carbonization. Examples thereof include a compound consisting of any one selected from a hydrogen derivative, a condensed polycyclic hydrocarbon derivative, a cyclic heterohydrocarbon derivative, and a condensed heteropolycyclic hydrocarbon derivative. Furthermore, the compound etc. which are represented by Chemical formula 4-Chemical formula 13 ([Chemical Formula 4]-[Chemical Formula 13]) are mentioned.
本発明の「抗ＨＩＶ剤」とは、抗ＨＩＶ活性を有するグルコシダーゼ活性阻害用組成物を有効成分として含む剤であればいずれの剤も含むことができる。本発明の抗ＨＩＶ活性を有するグルコシダーゼ活性阻害用組成物のみからなる剤であってもよく、また、薬物学的に許容される担体を含むものであってもよい。 The “anti-HIV agent” of the present invention can include any agent as long as it is an agent containing a composition for inhibiting glucosidase activity having anti-HIV activity as an active ingredient. It may be an agent consisting only of the composition for inhibiting glucosidase activity having anti-HIV activity of the present invention, or may contain a pharmaceutically acceptable carrier.
本発明の「グルコシダーゼ活性阻害用組成物」は、ｉｎ ｓｉｌｉｃｏバーチャルスクリーニングを行うことによってスクリーニングされることは好ましい。このスクリーニングには、次のようなスクリーニングルールを用いることができる。
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位のタンパク質表面を、半径１〜２オングストロームの球を用いて覆う。
２）活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、化合物とタンパク質の疎水性相互作用を生じる可能性空間とする。さらに、活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、リガンドとタンパク質の親水性相互作用を生じる可能性空間とする。
３）活性部位内に設置した可能性空間に少なくとも１〜５以上の原子が存在し、かつ、活性部位を逸脱しない化合物の検索を行う。
The “composition for inhibiting glucosidase activity” of the present invention is preferably screened by performing in silico virtual screening. The following screening rules can be used for this screening.
1) The protein surface of the active site estimated with respect to the X-ray crystallographic image of glucosidase is covered with a sphere having a radius of 1 to 2 angstroms.
2) 1 to 10 spheres having a radius of 1 to 2 angstroms are placed in the active site to form a space where a hydrophobic interaction between a compound and a protein may occur. Furthermore, 1 to 10 spheres having a radius of 1 to 2 angstroms are placed in the active site to create a space where a hydrophilic interaction between the ligand and the protein may occur.
3) Search for compounds that have at least 1 to 5 atoms in the possibility space placed in the active site and do not deviate from the active site.
さらに、本発明の「グルコシダーゼ活性阻害用組成物」のスクリーニングには、次のようなスクリーニングルールを用いることができる。
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位空間に対して、１つの化合物当り１０〜１００００のコンフォマーを作成し、活性部位空間に化合物を配置する。
２）得られたモデルを水素結合、配位結合、イオン的相互作用、化合物自由度、脱溶媒和エネルギー、化合物の埋没度、物理的な接触及び化合物の基質結合部位に対する充填率について、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアのスコアリング関数によって評価し、上位１／３をヒット化合物とする。
３）上記２）で次に、得られた化合物の群に対し、水素結合、配位結合、イオン的相互作用を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする。
４）さらに、上記３）で得られた化合物の群に対し、水素結合を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする。
Furthermore, the following screening rules can be used for screening the “composition for inhibiting glucosidase activity” of the present invention.
1) For the active site space estimated from the X-ray crystallographic image of glucosidase, 10 to 10,000 conformers per compound are prepared, and the compounds are arranged in the active site space.
2) The chemical model of the obtained model for hydrogen bonds, coordination bonds, ionic interactions, compound degrees of freedom, desolvation energy, compound embedment, physical contact and filling rate of the compound to the substrate binding site Group Inc. The scoring function of the MOE software is used, and the top 1/3 is regarded as a hit compound.
3) Next, in the above 2), the group of compounds obtained is screened with an emphasis on hydrogen bonds, coordination bonds, and ionic interactions, and evaluated by the same scoring function. Is a hit compound.
4) Further, the compound group obtained in the above 3) is screened with an emphasis on hydrogen bonding, evaluated by the same scoring function, and the top 1/3 is regarded as a hit compound.
さらに、本発明の「グルコシダーゼ活性阻害用組成物」のスクリーニングには、グルコシダーゼ阻害活性をバイオアッセイによってスクリーニングしてもよい。 Furthermore, the “glucosidase activity inhibiting composition” of the present invention may be screened for glucosidase inhibitory activity by a bioassay.
以下、実施例をあげて本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
グルコシダーゼ活性阻害用組成物のスクリーニング方法
１．１次スクリーニング
ｉｎ ｓｉｌｉｃｏバーチャルスクリーニングを、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアを用いて以下の１次スクリーニングルールに従い行った。
標的酵素となるグルコシダーゼの立体構造座標を用いて、基質が結合可能な部位を探索し、基質の分子サイズに適合するくぼみを探索し、得られた最適なくぼみ空間を基質結合部位と決定した。得られた基質結合部位を解析し基質結合部位の形状を抽出し基質結合部位モデルを作成した。そのモデルに対して、阻害剤候補化合物を当てはめ基質結合部位よりも大きな分子を排除した。
Screening Method for Composition for Inhibiting Glucosidase Activity 1.1 Primary Screening In silico virtual screening was performed by Chemical Computing Group Inc. The following primary screening rules were performed using MOE software.
Using the three-dimensional structure coordinates of glucosidase as the target enzyme, a site where the substrate can be bound was searched, a recess suitable for the molecular size of the substrate was searched, and the obtained optimal recess space was determined as the substrate binding site. The obtained substrate binding site was analyzed, the shape of the substrate binding site was extracted, and a substrate binding site model was created. For that model, candidate inhibitor compounds were fitted to exclude molecules larger than the substrate binding site.
＜１次スクリーニングルール＞
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位のタンパク質表面を、半径１〜２オングストロームの球を用いて仮想的に覆った。
２）活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、化合物とタンパク質の疎水性相互作用を生じる可能性空間とした。
３）さらに、活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、リガンドとタンパク質の親水性相互作用を生じる可能性空間として、紫色のメッシュの玉で示した（図１）
４）活性部位内に設置した可能性空間に少なくとも１〜５以上の原子が存在し、かつ、活性部位を逸脱しない化合物の検索を行った。
<Primary screening rules>
1) The protein surface of the active site estimated from the X-ray crystallographic image of glucosidase was virtually covered with a sphere having a radius of 1 to 2 angstroms.
2) 1 to 10 spheres having a radius of 1 to 2 angstroms were placed in the active site to create a potential space for causing a hydrophobic interaction between the compound and the protein.
3) Furthermore, 1 to 10 spheres having a radius of 1 to 2 angstroms were placed in the active site, and a purple mesh ball was shown as a potential space for generating a hydrophilic interaction between the ligand and the protein (FIG. 1).
4) A search was made for compounds that had at least 1 to 5 atoms in the possibility space placed in the active site and did not deviate from the active site.
この１次スクリーニングにより、６，０００，０００構造の化合物データベース“Ａｎ ａｄａｐｔａｔｉｏｎ ｆｏｒ ｔｈｅ Ｌｉｐｉｎｓｋｉ’ｓ Ｒｕｌｅ ｏｆ ｆｉｖｅ”から、スクリーニングルールに当てはまる２８，４４１個の化合物を得た。 By this primary screening, 28,441 compounds satisfying the screening rules were obtained from the compound database “An adaptation for the Lipinski's Rule of five” having a structure of 6,000,000.
２．２次スクリーニング
１次スクリーニングと同様に、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアを用いて、以下の２次スクリーニングルールに従い、ｉｎ ｓｉｌｉｃｏバーチャルスクリーニングを行った。
2.2 Secondary Screening Similar to the primary screening, Chemical Computing Group Inc. In silico virtual screening was performed using MOE software according to the following secondary screening rules.
＜２次スクリーニングルール＞
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位空間に対して、１つの化合物当り１０〜１００００のコンフォマーを仮想的に作成し、活性部位空間に化合物を配置した。
２）得られたモデルの水素結合、配位結合、イオン的相互作用、化合物自由度、脱溶媒和エネルギー、化合物の埋没度、物理的な接触及び化合物の基質結合部位に対する充填率について、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアのスコアリング関数によって評価し、上位１／３をヒット化合物とした。
３）上記２）で得られた化合物の群に対し、水素結合、配位結合、イオン的相互作用を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とした。
４）さらに、上記３）で得られた化合物の群に対し、水素結合を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とした。
これにより、計算精度を高め、化合物数をしぼることができた。
<Secondary screening rules>
1) With respect to the active site space estimated from the X-ray crystallographic image of glucosidase, 10 to 10,000 conformers per compound were virtually created, and the compounds were arranged in the active site space.
2) Regarding the hydrogen bond, coordination bond, ionic interaction, compound degree of freedom, desolvation energy, compound embedment, physical contact, and packing ratio of the compound to the substrate binding site of the obtained model, Chemical Computing Group Inc. The scoring function of the MOE software was evaluated and the top 1/3 was regarded as a hit compound.
3) The group of compounds obtained in 2) above is screened with an emphasis on hydrogen bonds, coordination bonds, and ionic interactions, and evaluated by the same scoring function. It was.
4) Further, the compound group obtained in 3) above was screened with an emphasis on hydrogen bonding, evaluated by the same scoring function, and the top 1/3 was regarded as a hit compound.
Thereby, the calculation accuracy was improved and the number of compounds could be reduced.
この２次スクリーニングにより、１次スクリーニングによって得られた２８，４４１個の化合物から、スクリーニングルールに当てはまる２個の化合物を得た。これらの化合物の構造を化学式２（下記、［化２］）及び化学式３（下記、［化３］）として示した。 By this secondary screening, two compounds that meet the screening rules were obtained from 28,441 compounds obtained by the primary screening. The structures of these compounds are shown as Chemical Formula 2 (below, [Chemical Formula 2]) and Chemical Formula 3 (below, [Chemical Formula 3]).
［化２］
[Chemical formula 2]
［化３］
[Chemical formula 3]
３．３次スクリーニング
２次スクリーニングで得られた２個の化合物のグルコシダーゼ阻害活性をバイオアッセイによりスクリーニングした。
3. Secondary screening The glucosidase inhibitory activity of the two compounds obtained in the secondary screening was screened by a bioassay.
１）ｉｎ ｖｉｔｒｏ アッセイ
酵素反応は次のように行った。
各化合物（いずれも純度９９％以上）について、１０ｍＭジメチルスルフォキシド溶液を作製した。この溶液をジメチルスルフォキシドで希釈し、各化合物を７８μＭ〜１０ｍＭ含む化合物溶液を得た。
各濃度の化合物溶液５μＬをそれぞれ用い、１００ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０）２０μＬ、グルコシダーゼ溶液２５μＬとともに３７℃・２０分・６００ｒｐｍで振盪しプレインキュベートした。
その後、これに基質溶液（３ｍＭ ｐ−Ｎｉｔｒｏｐｈｅｎｙｌ α‐Ｄ‐ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ／１００ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０））２５μＬを加え、３７℃・２０分・６００ｒｐｍで振盪し酵素反応を行い、０．３Ｍ Ｋ2ＣＯ3 ７５μＬで停止した。各化合物におけるグルコシダーゼ阻害活性は、反応により遊離したパラニトロフェノールの４０５ｎｍにおける吸光度を測定することにより求めた。
1) In vitro assay The enzyme reaction was performed as follows.
A 10 mM dimethyl sulfoxide solution was prepared for each compound (both having a purity of 99% or more). This solution was diluted with dimethyl sulfoxide to obtain a compound solution containing 78 μM to 10 mM of each compound.
Using 5 μL of the compound solution of each concentration, the mixture was shaken at 37 ° C., 20 minutes, 600 rpm with 20 μL of 100 mM sodium phosphate buffer (pH 7.0) and 25 μL of the glucosidase solution, and preincubated.
Thereafter, 25 μL of a substrate solution (3 mM p-Nitrophenyl α-D-glucopyranoside / 100 mM sodium phosphate buffer (pH 7.0)) was added thereto, and the mixture was shaken at 37 ° C. for 20 minutes at 600 rpm to carry out an enzyme reaction. Stopped with 75 μL of 3M K 2 CO 3 . The glucosidase inhibitory activity of each compound was determined by measuring the absorbance at 405 nm of paranitrophenol released by the reaction.
その結果、この２個の化合物のうち、化学式２で表される化合物がグルコシダーゼ阻害活性を有することが確認され、この化合物を本発明のグルコシダーゼ活性阻害用組成物とした。 As a result, of these two compounds, it was confirmed that the compound represented by Chemical Formula 2 has glucosidase inhibitory activity, and this compound was used as the composition for inhibiting glucosidase activity of the present invention.
また、上記で得られたグルコシダーゼ活性阻害用組成物と同じ構造（上記、式［化１］で表される構造）を母核として有する化合物（化学式４〜化学式１３（下記、式［化４］〜［化１３］で表される化合物））をナミキ商事株式会社より購入し、本発明のグルコシダーゼ活性阻害用組成物とした。
さらに、図２において、化学式７又は化学式１０で表される化合物がグルコシダーゼ活性阻害用組成物として、どのようにグルコシダーゼに結合するかを結合モデルで示した。
In addition, compounds having the same structure as the glucosidase activity inhibition composition obtained above (the structure represented by the above formula [Chemical Formula 1]) as a parent nucleus (Chemical Formula 4 to Chemical Formula 13 (the following Formula [Chemical Formula 4]). To [Chemical Formula 13])) was purchased from Namiki Shoji Co., Ltd. and used as the composition for inhibiting glucosidase activity of the present invention.
Furthermore, in FIG. 2, how the compound represented by Chemical Formula 7 or Chemical Formula 10 binds to glucosidase as a composition for inhibiting glucosidase activity is shown by a binding model.
［化４］
[Chemical formula 4]
［化５］
[Chemical formula 5]
［化６］
[Chemical 6]
［化７］
[Chemical 7]
［化８］
[Chemical 8]
［化９］
[Chemical 9]
［化１０］
[Chemical Formula 10]
［化１１］
[Chemical 11]
［化１２］
[Chemical 12]
［化１３］
[Chemical 13]
２）Ｃｅｌｌ ｂａｓｅ アッセイ
上記で得られたグルコシダーゼ活性阻害用組成物を用い、ＨＩＶ感染細胞に対する効果を調べた。
即ち、上記で得られた各グルコシダーゼ活性阻害用組成物をＤＭＳＯに溶解し、１００ｍＭとしたものをそれぞれＨＩＶ感染細胞に付与し、Ｃｅｌｌ ｂａｓｅアッセイによって５０％細胞死阻止濃度（ＥＣ５０）及び細胞毒性濃度（ＣＣ５０）を調べ、治療係数ＳＩ値（ＳＩ値＝ＣＣ５０値／ＥＣ５０値）を求めた。
ＨＩＶ感染細胞は、ＦＣＳを含むＤＭＥＭ培地に懸濁し、マイクロプレートに播種したヒトＴ細胞株に、常法により作製した感染性ＨＩＶ−１（ＮＬ４３２株）を感染させ、３７℃で培養することで得たものを用いた。
また、各化合物によるＨＩＶ感染細胞の増殖阻害効果は、ＨＩＶ感染細胞を培養した後、培養上清中のＨＩＶ−１逆転写酵素量を、ポリＡＲＮＡを鋳型とする逆転写酵素アッセイにより定量することで評価した。さらに、細胞毒性をＷＳＴ法（ＷＳＴアッセイキット）により評価した。
比較として、ナミキ商事株式会社より購入した化学式１４（下記、式［化１４］）で表される化合物を用いて、５０％細胞死阻止濃度（ＥＣ５０）及び細胞毒性濃度（ＣＣ５０）を調べ、治療係数ＳＩ値（ＳＩ値＝ＣＣ５０値／ＥＣ５０値）を同様に求めた。
その結果、表１に示したように、本発明のグルコシダーゼ活性阻害用組成物は、治療係数ＳＩ値が抗ＨＩＶ活性を有する公知の化合物より高く、強い抗ＨＩＶ活性を有することが確認された。
2) Cell base assay Using the composition for inhibiting glucosidase activity obtained above, the effect on HIV-infected cells was examined.
That is, each glucosidase activity inhibiting composition obtained above was dissolved in DMSO and applied to HIV-infected cells, and 50% cell death inhibitory concentration (EC50) and cytotoxic concentration were determined by Cell base assay. (CC50) was examined, and the therapeutic index SI value (SI value = CC50 value / EC50 value) was determined.
HIV-infected cells are suspended in DMEM medium containing FCS, and infected with infectious HIV-1 (NL432 strain) prepared by a conventional method in a human T cell line seeded on a microplate, and cultured at 37 ° C. What was obtained was used.
The growth inhibitory effect of HIV-infected cells by each compound is determined by quantifying the amount of HIV-1 reverse transcriptase in the culture supernatant by reverse transcriptase assay using polyARNA as a template after culturing HIV-infected cells. It was evaluated with. Furthermore, cytotoxicity was evaluated by the WST method (WST assay kit).
As a comparison, a 50% cell death inhibitory concentration (EC50) and a cytotoxic concentration (CC50) were examined using a compound represented by chemical formula 14 (below, formula [Chemical Formula 14]) purchased from Namiki Shoji Co., Ltd. The coefficient SI value (SI value = CC50 value / EC50 value) was similarly determined.
As a result, as shown in Table 1, it was confirmed that the composition for inhibiting glucosidase activity of the present invention has a therapeutic index SI value higher than that of known compounds having anti-HIV activity and has strong anti-HIV activity.
［化１４］
[Chemical 14]
本発明のグルコシダーゼ活性阻害用組成物は、糖鎖を制御できる優れたグルコシダーゼ活性阻害用組成物であり、糖鎖生物学における研究用試薬等に用いることができる。また、ヒト等の摂取において安全性が高く、抗ＨＩＶ剤の有効成分として用いることができる。 The composition for inhibiting glucosidase activity of the present invention is an excellent composition for inhibiting glucosidase activity capable of controlling sugar chains, and can be used as a research reagent in sugar chain biology. In addition, it is highly safe for human consumption and can be used as an active ingredient of anti-HIV agents.
Claims (8)
［化１］
A composition for inhibiting glucosidase activity, which is a compound having a structure represented by the following formula [Chemical Formula 1] as a mother nucleus.
[Chemical 1]
［化４］
［化５］
［化６］
［化７］
［化８］
［化９］
［化１０］
［化１１］
［化１２］
［化１３］
The composition for inhibiting glucosidase activity according to any one of claims 1 to 3, which is a compound represented by any one of the following formulas [Chemical Formula 4] to [Chemical Formula 13].
[Chemical formula 4]
[Chemical formula 5]
[Chemical 6]
[Chemical 7]
[Chemical 8]
[Chemical 9]
[Chemical Formula 10]
[Chemical 11]
[Chemical 12]
[Chemical 13]
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位のタンパク質表面を、半径１〜２オングストロームの球を用いて仮想的に覆う
２）活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、化合物とタンパク質の疎水性相互作用を生じる可能性空間とする
３）さらに、活性部位内に半径１〜２オングストロームの球を１〜１０個設置し、リガンドとタンパク質の親水性相互作用を生じる可能性空間とする
４）活性部位内に設置した可能性空間に少なくとも１〜５以上の原子が存在し、かつ、活性部位を逸脱しない化合物の検索を行う The screening method of the composition for glucosidase activity inhibition by performing in silico virtual screening by the following screening rules.
1) The protein surface of the active site estimated from the X-ray crystallographic image of glucosidase is virtually covered with a sphere with a radius of 1 to 2 angstroms. 2) A sphere with a radius of 1 to 2 angstroms is placed in the active site. Place 1-10 spheres to generate a hydrophobic interaction between the compound and protein. 3) Furthermore, place 1-10 spheres with a radius of 1-2 angstroms in the active site to make the hydrophilicity of the ligand and protein. 4) Search for compounds that have at least 1 to 5 atoms in the possibility space placed in the active site and do not deviate from the active site.
１）グルコシダーゼのＸ線結晶解析像に対して推定される活性部位空間に対して、１つの化合物当り１０〜１００００のコンフォマーを仮想的に作成し、活性部位空間に化合物を配置する
２）得られたモデルを水素結合、配位結合、イオン的相互作用、化合物自由度、脱溶媒和エネルギー、化合物の埋没度、物理的な接触及び化合物の基質結合部位に対する充填率について、Ｃｈｅｍｉｃａｌ Ｃｏｍｐｕｔｉｎｇ Ｇｒｏｕｐ Ｉｎｃ．のＭＯＥソフトウエアのスコアリング関数によって評価し、上位１／３をヒット化合物とする
３）上記２）で次に、得られた化合物の群に対し、水素結合、配位結合、イオン的相互作用を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする
４）さらに、上記３）で得られた化合物の群に対し、水素結合を重要視したスクリーニングを行い、同様のスコアリング関数によって評価し、上位１／３をヒット化合物とする Furthermore, the screening method of the composition for glucosidase activity inhibition of Claim 6 which has glucosidase inhibitory activity by performing in silico virtual screening by the following screening rules.
1) Virtually create 10-10000 conformers per compound for the active site space estimated for the X-ray crystallographic image of glucosidase, and place the compound in the active site space 2) The Chemical Computation Group Inc. was used for hydrogen bonds, coordination bonds, ionic interactions, compound degrees of freedom, desolvation energy, compound embedment, physical contact, and packing ratio of the compound to the substrate binding site. 3) Using the top 1/3 as a hit compound, 3) Next, in 2) above, hydrogen bonds, coordination bonds, and ionic interactions are obtained. Is evaluated by the same scoring function, and the top 1/3 is regarded as a hit compound. 4) Further, for the group of compounds obtained in the above 3), the hydrogen bond is emphasized. And evaluate with the same scoring function, and the top 1/3 is regarded as a hit compound
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| US10913716B2 (en) | 2016-03-31 | 2021-02-09 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11180450B2 (en) | 2016-04-01 | 2021-11-23 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11083707B2 (en) | 2017-03-31 | 2021-08-10 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11179368B2 (en) | 2017-03-31 | 2021-11-23 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11053196B2 (en) | 2017-05-22 | 2021-07-06 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11407715B2 (en) | 2017-05-22 | 2022-08-09 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11702387B2 (en) | 2017-05-22 | 2023-07-18 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
| US11795149B2 (en) | 2017-05-22 | 2023-10-24 | Janssen Pharmaceuticals, Inc. | Substituted indoline derivatives as dengue viral replication inhibitors |
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