JP6304675B2 - Screening method for carbonic anhydrase inhibitors - Google Patents
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、創薬効果的プローブとして機能性アミノ酸ヒスチジンの挙動を利用する炭酸脱水酵素阻害剤のNMRスクリーニング法(Histidine-based NMRスクリーニング法)に関する。 The present invention relates to an NMR screening method for a carbonic anhydrase inhibitor (Histidine-based NMR screening method) using the behavior of a functional amino acid histidine as a drug discovery effective probe.
NMR（核磁気共鳴）法は、溶液状態にある蛋白質の構造解析法として構造と機能の相関を得る上で欠かせない存在である。中でも、安定同位体標識法や分子生物学的遺伝子操作手法の発展に伴い、現在では1〜2万程度の比較的小さい分子量の蛋白質の立体構造決定法として既に確立されている。
しかし、蛋白質の立体構造を基盤として合理的な治療薬設計(Rational Drug Design)を目指す分野において、ターゲットとなる蛋白質の構造を決定する手段としてNMR法が使用されることは珍しく、むしろリガンド結合による局所構造の変化に関する情報を取得する手段として使われている。特に、1996年に「Structure-activity Relationships(SAR)
by NMR」という名前で初めて報告された低分子フラグメントを連結するスクリーニング戦略が脚光を浴び、最近ではFragment
Based NMR Screening(FBNS)又はFragment Based Drug
Descovery(FBDD)としてよく知られている。
The NMR (nuclear magnetic resonance) method is indispensable for obtaining a correlation between structure and function as a structural analysis method of a protein in a solution state. In particular, with the development of stable isotope labeling methods and molecular biological genetic manipulation techniques, it has already been established as a method for determining the three-dimensional structure of proteins with a relatively small molecular weight of about 10,000 to 20,000.
However, in the field aiming at rational drug design based on the three-dimensional structure of protein, it is rare that NMR method is used as a means to determine the structure of target protein, rather it is based on ligand binding. It is used as a means to acquire information about changes in local structure. In particular, in 1996, `` Structure-activity Relationships (SAR)
The screening strategy for linking small-molecule fragments, first reported under the name `` by NMR '', came into the limelight, and recently Fragment
Based NMR Screening (FBNS) or Fragment Based Drug
Well known as Descovery (FBDD).
これらの手法は、生体分子ターゲットの構造知識を基盤として新たな治療薬物を発見するものである。そこでは、1)ケミカルライブラリーに登録された化合物の数より遙かに少ない低分子化合物群の中から、ターゲット蛋白質と相互作用する化合物(分子フラグメント)を選出すること、2)蛋白質の異なる位置に相互作用する2つの分子フラグメントの方向性をバイオインフォマティクス解析、距離情報解析、相互作用解析法を用いて決定すること、3)分子フラグメントを修飾・連結することによって、より高い結合能をもつリード化合物を見出すことが行われ、この手法によりスクリーニングに要する手間は実質的に10-4程度になるとの試算が報告されている。
NMR法はこれら3つのプロセスのうち主にa)15N-1H
HSQC法（15N核-1H核相関Heteronuclear Single
Quantum Coherence）を用いて分子フラグメント結合による蛋白質アミドシグナルの変化を検出すること(相互作用の検出)、b)NOEに基づく距離・相対的配向情報を得ること等に用いられる。特にa)は一度の15N安定同位体標識蛋白質の調製によって省力的かつ安価に測定を行うことが可能であるため、簡便さに優れるという利点をもつ（例えば特許文献１〜３）。
These methods discover new therapeutic drugs based on the structural knowledge of biomolecular targets. There, 1) select a compound (molecular fragment) that interacts with the target protein from a group of low molecular compounds that is much smaller than the number of compounds registered in the chemical library, and 2) different positions of the protein. Determine the direction of two molecular fragments that interact with each other using bioinformatics analysis, distance information analysis, and interaction analysis methods, and 3) lead with higher binding ability by modifying and linking molecular fragments It has been reported that a compound is found, and that the labor required for screening by this technique is substantially about 10 −4 .
NMR methods are mainly a) 15 N- 1 H of these three processes.
HSQC method ( 15 N nucleus- 1 H nucleus correlation Heteronuclear Single
Quantum Coherence) is used to detect changes in protein amide signals due to molecular fragment binding (detection of interactions), b) to obtain distance / relative orientation information based on NOE, etc. In particular, a) has the advantage of being excellent in simplicity because it can be measured labor-saving and inexpensively by preparing a 15 N stable isotope-labeled protein once (for example,
しかし同時に、A)分子フラグメントが相互作用する位置は側鎖であることが多いために、その結合に伴って変化するアミドシグナルの移動度は低いこと、B)芳香環位置・向きの僅かなずれといった局所構造の変化やpH変化、イオン強度変化によって分子フラグメント結合と相対的に同程度に移動したおびただしい数のシグナルの中から、正確なフラグメント結合を示すシグナルをピンポイントに特定することが難しいこと、C)CA（Carbonic Anhydrase：炭酸脱水酵素）のように29.3 k-Daの蛋白質となるとアミドシグナルの数は250を越えるためにさらにその特定を著しく困難なものにすること、といったNMR特有の問題にその発展が阻まれてきた。
また、炭酸脱水酵素阻害効果を有する利尿剤等に含まれるスルフォンアミド基は過敏症発生の問題が指摘されているため、スルフォンアミド基を含まない新規阻害剤の開発が期待されている。
また、炭酸脱水酵素阻害剤の開発手法として、水素イオンの初期濃度変化を測定するpH指示薬法が知られているが、濃度変化を正確に測定することが困難という問題がある。
However, at the same time, A) the position where the molecular fragment interacts is often a side chain, so the mobility of the amide signal that changes with the binding is low, and B) a slight shift in the position and orientation of the aromatic ring. It is difficult to pinpoint the signal that indicates the exact fragment binding among the numerous signals that have moved to the same extent as the molecular fragment binding due to local structural changes, pH changes, and ionic strength changes. , C) NMR-specific problems, such as CA (Carbonic Anhydrase), 29.3 k-Da protein, the number of amide signals exceeds 250, making its identification extremely difficult Its development has been hindered.
In addition, since the sulfonamide group contained in diuretics having a carbonic anhydrase inhibitory effect has been pointed out to cause hypersensitivity, development of a novel inhibitor not containing a sulfonamide group is expected.
Further, as a method for developing a carbonic anhydrase inhibitor, a pH indicator method for measuring a change in the initial concentration of hydrogen ions is known, but there is a problem that it is difficult to accurately measure the change in concentration.
本発明は、創薬効果的プローブとして機能性アミノ酸ヒスチジンの挙動を利用する炭酸脱水酵素阻害剤のNMRスクリーニング法(Histidine-based NMRスクリーニング法)を提供することを目的とする。 An object of the present invention is to provide an NMR screening method for a carbonic anhydrase inhibitor (Histidine-based NMR screening method) using the behavior of a functional amino acid histidine as a drug discovery effective probe.
本願発明者は、活性部位に非常によくみられる機能性アミノ酸ヒスチジンに焦点を絞り、これまでの自身の研究において開発したヒスチジンNMR解析手法をSAR/FBNS/FBDD手法と組み合わせることによって、ヒスチジンに特化した新たなNMRスクリーニング手法を開発することが可能であると着想した。そして、ヒスチジンを活性部位に含むCAにこれを適用することによって、より合理的かつ迅速に利尿・高血圧治療薬を設計することを目的として研究を行い、本発明を完成させた。 The inventor focused on the functional amino acid histidine that is very common in the active site, and combined the histidine NMR analysis method developed in his own research with the SAR / FBNS / FBDD method. I thought that it was possible to develop a new NMR screening method. Then, by applying this to CA containing histidine in the active site, research was conducted for the purpose of designing a therapeutic drug for diuresis and hypertension more rationally and rapidly, and the present invention was completed.
本発明の炭酸脱水酵素阻害剤のスクリーニング方法は、ヒト由来炭酸脱水酵素II型(hCAII)のうち亜鉛配位に係るヒスチジン（His94、His96及びHis119）のイミダゾール側鎖(窒素核及び水素核)に由来するNMRシグナルのpH依存性（亜鉛結合水のイオン化に係る情報）と、His64の酸解離定数pKa及び互変異性平衡定数KTを指標として用いることを特徴とする。
また、被験物質添加時のNMRスペクトルによって観察されたシグナルのケミカルシフトがpH非依存的になった場合に、亜鉛配位に係るヒスチジンと当該被験物質とが結合していると判断することを特徴とする。
また、炭酸脱水酵素単独時のHis64の酸解離定数pKa及び互変異性平衡定数KTと、被験物質添加時のHis64の酸解離定数pKa及び互変異性平衡定数KTとを比較して、少なくともいずれか一方の値が変化した場合(pKa≠7.2またはKT≠1.0、精度５％を加味した有意な変化の場合)に、当該被験物質が炭酸脱水酵素に結合している又は相互作用していると判断することを特徴とする。
The screening method of the carbonic anhydrase inhibitors of the present invention, human carbonic anhydrase II type (HCAII) histidine according to zinc coordination among (His94, His96 and His 119) the imidazole side chain of (nitrogen nuclei and hydrogen nuclei) the pH dependence of the NMR signals due (information relating to the ionization of zinc bound water) to, is characterized by using as an index the acid dissociation constant pK a and tautomeric equilibrium constant K T of His64.
In addition, when the chemical shift of the signal observed by the NMR spectrum at the time of adding the test substance becomes pH-independent, it is judged that the test substance is bound to the histidine related to zinc coordination. And
In comparison with carbonic anhydrase alone acid dissociation constant pK a and tautomeric equilibrium constant K T of the His64 of time, the acid dissociation constant of His64 during the test substance added pK a and tautomeric equilibrium constant K T , If at least one of the values changes (if pK a ≠ 7.2 or K T ≠ 1.0, significant change taking into
また、前記NMRスペクトルが、15N標識した炭酸脱水酵素の２次元15N-1H NMR相関スペクトルであることを特徴とする。 Further, the NMR spectrum is a two-dimensional 15 N- 1 H NMR correlation spectrum of 15 N-labeled carbonic anhydrase.
蛋白質構造・ゲノムプロジェクトが終了し、それらの情報を基盤として1)創薬や2)環境科学といったライフサイエンスに実際に貢献する手法の開発を行うことが喫緊の課題である。
本願発明者は、1)についてアプローチするために、蛋白質の機能の発現にヒスチジンが多く見られることに着眼し、蛋白質中に分散的に存在するその挙動を追うことによって、これまでのNMRスクリーニング手法にかかる曖昧さを低減した「創薬効果的プローブとして機能性アミノ酸ヒスチジンの挙動を利用するNMRスクリーニング法(Histidine-based NMRスクリーニング法)」の開発が可能であることを着想した。
そして、ヒスチジンが機能発現に関わる蛋白質系において、誰もが容易に使うことが可能な研究手法を整理し、これまで1サンプルあたり数ヶ月以上かかっていた構造基盤型薬設計-スクリーニング手法を数時間から5日程度で行う本願発明の技術を開発した。
After completing the Protein Structure / Genome Project, it is an urgent task to develop methods that actually contribute to life science such as 1) drug discovery and 2) environmental science based on such information.
In order to approach 1), the inventor of the present application pays attention to the fact that many histidines are seen in the expression of protein functions, and by following the behavior that exists dispersively in proteins, conventional NMR screening methods The idea was that it was possible to develop an "NMR screening method that uses the behavior of functional amino acid histidine as an effective drug discovery probe (Histidine-based NMR screening method)".
In the protein system in which histidine is involved in functional expression, the research methods that anyone can easily use are organized, and the structure-based drug design-screening method, which previously took several months or more per sample, takes several hours. The technology of the present invention was developed in about 5 days.
本願発明は、特に教科書レベルでみられる炭酸脱水酵素を例として用い本手法によるより合理的な新薬設計に取組むことによって利尿・降圧作用をもつ新薬の具体的創出を行い、最終的にヒスチジンが活性に関与する蛋白質全般に適用可能かつ簡便な新規スクリーニング技術に関する。
ヒスチジンを用いてこのようなスクリーニング手法が可能であることから、他のアミノ酸もまた同様に開発されることが可能であり、最終的にアミノ酸側鎖網羅型のスクリーニング手法の開発が視野に入る。
The present invention specifically creates a new drug with diuretic and antihypertensive action by working on more rational new drug design by this method using carbonic anhydrase found especially at textbook level as an example, and finally histidine is active The present invention relates to a novel screening technique that can be applied to all proteins involved in.
Since such a screening method is possible using histidine, other amino acids can be developed in the same manner, and finally, development of a screening method that covers amino acid side chains is in the field of view.
一方、2)についてアプローチするために、本研究で解明されるCAの触媒機構からその触媒必須因子を抽出、かつ複数の官能基を人工的に適宜配置することによって、酵素を模倣した二酸化炭素水和触媒を開発することが可能である。その機能的な配置は、ヒスチジンの挙動によって検出・確認することが可能であるため、ここでもまた申請者が見出した経験則を基盤とする「Histidine-based NMRスクリーニング法」によって探索することが可能である。
本研究をこのような触媒低分子化の方向に進めるならば、二酸化炭素水和技術によって、植物が光合成前段階で行う大気中二酸化炭素の取込みを模倣するバイオミメティックスがさらに現実味を帯びとなり、環境分野の課題解決に大きくアプローチすることが可能である。本発明は、生体分子の相互作用解明にかかる真の構造生命科学において構築されるスクリーニング技術を基盤とすることで、合理的創薬と二酸化炭素回収という2つの大きなライフサイエンス課題を解決に導く技術である。
On the other hand, in order to approach 2), carbon dioxide water that mimics the enzyme is extracted by extracting the essential factors of the catalyst from the catalytic mechanism of CA elucidated in this study and artificially arranging multiple functional groups. It is possible to develop sum catalysts. Its functional arrangement can be detected and confirmed by the behavior of histidine, so it can be searched here again by the “Histidine-based NMR screening method” based on the empirical rule found by the applicant. It is.
If this research is advanced in the direction of such a low molecular weight catalyst, biomimetics that mimic the uptake of atmospheric carbon dioxide by plants in the pre-photosynthesis stage will become more realistic with the carbon dioxide hydration technology, and environmental fields will become more realistic. It is possible to approach a large number of issues. The present invention is based on the screening technology built in the true structural life science for elucidating the interaction of biomolecules, and this technology leads to solving two major life science issues: rational drug discovery and carbon dioxide recovery. It is.
［本願発明と従来手法との比較］
発明者は、これまでの20年間の殆どを、NMR法を用いた酵素ヒスチジンの挙動解析に費やし、機能発現時にみられるヒスチジンの酸塩基平衡、互変異性平衡、回転、金属配位といった様々な化学特性がアミノ酸変異等によってどのような影響を受けるか又はどのようなシグナル変化を示すかを調べてきた。
その研究で発明者が開発したヒスチジン解析手法とSAR/FBNS/FBDD手法とを組み合わせてなる本発明のヒスチジンに特化した新たなNMRスクリーニング手法と従来手法との比較を図１に示す。
[Comparison of the present invention and conventional methods]
The inventor has spent most of the past 20 years on the analysis of the behavior of the enzyme histidine using NMR, and has found various histidine acid-base balance, tautomeric equilibrium, rotation, metal coordination, etc. It has been investigated how chemical characteristics are affected by amino acid mutations and the like and what signal changes are exhibited.
FIG. 1 shows a comparison between a new NMR screening method specialized for histidine of the present invention, which is a combination of the histidine analysis method developed by the inventor in the research and the SAR / FBNS / FBDD method, and the conventional method.
図１（ａ）は従来手法である15N標識した炭酸脱水酵素（ここではヒト由来炭酸脱水酵素II型(hCAII)）のうちアミド領域の15N-1H HSQCスペクトルである。灰薄色は酵素単独、黒は降圧・利尿薬として知られるフロセミドとCAの複合体に由来するシグナルである。フロセミド添加によって移動したアミドシグナルの数はあまりに多く、その中から結合によって移動したシグナルを特定するには多大な労力と時間が必要であることがわかる。
一方、図１（ｂ）は本願発明である、同じく15N標識したヒト由来II型(hCAII)のうちイミダゾール領域の15N-1H HSQCスペクトルである。黒は酵素単独、灰薄色はフロセミド-CA複合体のシグナルである。
Ｌ字型で結ばれたシグナルが一つのヒスチジンのそれを示しており、矢印で示すように顕著に移動した数個のヒスチジンが一目でわかる。このスペクトルを得るための時間は、図１（ａ）に示した従来手法と同等かそれより短いので、従来のSAR/FBNS/FBDD手法が有する簡便さに優れるという利点を保ったまま、フラグメントと直接相互作用する側鎖のシグナルをピンポイントに捉えることが可能であることが分かる。
FIG. 1A is a 15 N- 1 H HSQC spectrum of the amide region of 15 N-labeled carbonic anhydrase (here, human-derived carbonic anhydrase type II (hCAII)), which is a conventional method. The light gray color is the enzyme alone, and the black signal is derived from the complex of furosemide and CA known as antihypertensive / diuretic. It can be seen that the number of amide signals transferred by the addition of furosemide is too large, and it takes a lot of labor and time to identify the signal transferred by binding.
Meanwhile, FIG. 1 (b) is a present invention, also a 15 N- 1 H HSQC spectrum of the imidazole region of 15 N-labeled human type II (hCAII). Black is the enzyme alone, light gray is the signal of furosemide-CA complex.
An L-shaped signal indicates that of one histidine, and several histidines that have moved significantly can be seen at a glance as indicated by arrows. Since the time for obtaining this spectrum is equal to or shorter than that of the conventional method shown in FIG. 1 (a), while maintaining the advantage of the convenience of the conventional SAR / FBNS / FBDD method, It can be seen that the signal of the side chain that directly interacts can be pinpointed.
次に、本発明に係るヒスチジン領域のNMRスペクトルを用いたスクリーニング法の効果を証明するべく、次の4つの項目について説明する。
1.ヒスチジンのシグナルから得られる情報
2.その情報が反映する構造・挙動(触媒機構)
3.阻害によって移動するシグナルが反映する構造・挙動(阻害機構)
4.これまでに報告されたCAを阻害する薬物の普遍的な相互作用原理
Next, the following four items will be described in order to prove the effect of the screening method using the NMR spectrum of the histidine region according to the present invention.
1. Information obtained from histidine signal
2. Structure and behavior reflected by the information (catalyst mechanism)
3.Structure / behavior reflected by signals that move due to inhibition (inhibition mechanism)
4. The universal interaction principle of drugs that have been reported so far
［1.ヒスチジンのシグナルから得られる情報(酸塩基平衡定数、互変異性平衡定数、金属配位)］
ヒスチジンのイミダゾリウムカチオンは２つの互変異性体(Nδ1-H異性体(1)とNε2-H異性体(3))と酸塩基平衡にある（図２）。そこではそれぞれとの平衡について微視的な定数K1、K2が与えられ、それらから巨視的な酸塩基平衡Ka(K1＋K2)と互変異性平衡定数KT(K1/K2)が得られる。ヒスチジンはこの３つの型の中でNMR時間軸上十分速く交換するため、観測されるケミカルシフトはその型の核の荷重平均で表される。イミダゾールを構成する1H、13C、15N核の中で、15Nのケミカルシフト変化はその交換に最も感受性が高く、水素が結合した15N核は167ppm付近に観測され、結合していないそれは250ppm付近に観測される。その差が82ppmあることから、他の環境変化に因るケミカルシフト差が2〜3ppmあるとしても、平衡定数の決定について5％に満たない誤差が保たれ、これは他の核と比較して精度が高い。
類似手法としてJ結合定数を用いた平衡定数決定手法が報告されているが、これと本手法を比較しても、簡便性と精度の両方について本手法の方が優れている。
イミダゾリウムカチオンのNδ1はおよそ179ppm、Nε2は181ppmに観測されることから、以下の式（数１）を用いてpHに依存した15N核シグナルの挙動をシュミレーションすると図３のようになる。
The imidazolium cation of histidine is in acid-base equilibrium with two tautomers (N δ1 -H isomer (1) and N ε2 -H isomer (3)) (Figure 2). Where microscopic constants K 1 for equilibrium with each, K 2 are given, they from macroscopic acid-base equilibrium K a (K 1 + K 2 ) and the tautomeric equilibrium constant K T (K 1 / K 2 ) is obtained. Since histidine exchanges fast enough on the NMR time axis among these three types, the observed chemical shift is expressed as a weighted average of that type of nucleus. Among the 1 H, 13 C, and 15 N nuclei that make up imidazole, the chemical shift change of 15 N is the most sensitive to the exchange, and the hydrogen-bonded 15 N nuclei are observed around 167 ppm and are not bonded. It is observed around 250 ppm. Since the difference is 82 ppm, even if the chemical shift difference due to other environmental changes is 2 to 3 ppm, an error of less than 5% is maintained in the determination of the equilibrium constant, compared to other nuclei. High accuracy.
Although an equilibrium constant determination method using J coupling constants has been reported as a similar method, even if this method is compared with this method, this method is superior in both simplicity and accuracy.
Since N δ1 of the imidazolium cation is observed at approximately 179 ppm and N ε2 is observed at 181 ppm, the following equation (Equation 1) is used to simulate the behavior of the 15 N nuclear signal depending on pH, as shown in FIG. .
実際に上記方法を活性部位にHis64と亜鉛結合ヒスチジンがみられる炭酸脱水酵素(CA)に適用した。その結果、図４に示すように1)酵素単独時のHis64のpKa,KTが特徴的であること(それぞれ7.2,1.0)、図５に示すように2)亜鉛結合水のpKaを示す亜鉛結合ヒスチジンの挙動がpH依存的であること、3)図４に示すように触媒能が維持されるY7F変異体においてHis64のKTが0.67に変化すること、4)図４に示すように阻害剤結合時は酵素単独と比べてHis64のpKa,KTが変化すること及び図５に示すように阻害剤結合時は亜鉛結合ヒスチジンの挙動がpH非依存的となることが判る。中でも4)は本酵素の阻害新薬スクリーニングだけでなく触媒模倣スクリーニングにも有用である。 In fact, the above method was applied to carbonic anhydrase (CA), which has His64 and zinc-binding histidine in the active site. As a result, the pK a of His64 of 1) when the enzyme alone as shown in FIG. 4, it K T is characteristic (respectively 7.2,1.0), the pK a of 2) zinc bound water as shown in FIG. 5 it behavior of zinc-binding histidine indicated is pH-dependent, 3) K T of His64 is changed to 0.67 in Y7F mutants catalytic ability is maintained as shown in FIG. 4, 4) as shown in FIG. 4 inhibitors during binding pK a of His64 as compared to the enzyme alone, at inhibitor binding as shown in and FIG. 5 K T is changed it can be seen that the behavior of the zinc-binding histidine is pH-independent on. Among them, 4) is useful not only for screening for new drugs that inhibit this enzyme, but also for screening for catalytic mimetics.
［2.その情報が反映する構造・挙動(触媒機構)］
NMR法によって得られるヒスチジン情報pKaとKTは、それぞれ環境の酸性度と水素結合相互作用を反映する数値である。特に、発明者はKTと水素結合相互作用の間に規則性があり(図６)、そこではδ1窒素と酸性基の間の水素結合相互作用が高まるとKTが上昇するという経験則があることを見出した。
これを上記1.の1)から3)に当てはめると、野生型、Y7F変異体のHis64のKT（1.0、0.7）は、酸性基と水素結合(HB)を形成したり切れたりすること(部分的にHB形成すること)を表していることが分かった。そして、これまで広く受け入れられているメカニズムと結晶構造にその変化を組み合わせて検討したところ、図７のような触媒機構が得られた。さらに量子化学的計算の結果と併せたところ、図８のようにHis64は２つの酸性基(亜鉛結合水とGlu106)と水素結合相互作用することで、His64のpKaとKTが決定される様子が露となり、妥当なエネルギーダイアグラムを作成するに至った。
[2. Structure and behavior reflected by the information (catalyst mechanism)]
Histidine information pK a and K T obtained by the NMR method are each numerical value that reflects the acidity and hydrogen bonding interactions environment. In particular, the inventor has regularity between KT and hydrogen bond interaction (FIG. 6), where the rule of thumb is that KT increases as the hydrogen bond interaction between δ1 nitrogen and acidic group increases. I found out.
When this is applied to 1) to 3) above, the wild-type, Y7F mutant His64 K T (1.0, 0.7) forms or breaks hydrogen bonds (HB) with acidic groups ( It was found that it partially represents HB formation). Then, when the change was combined with the widely accepted mechanism and the crystal structure, a catalyst mechanism as shown in FIG. 7 was obtained. It was further together with the results of quantum chemical calculation, His64 is by hydrogen bonding interactions with the two acidic groups (zinc binding water and Glu106), pK a and K T of His64 is determined as shown in FIG. 8 The situation became dew and a reasonable energy diagram was created.
［3.阻害によって移動するシグナルが反映する構造・挙動(阻害機構)］
阻害剤の添加によって亜鉛結合ヒスチジンの挙動がpH非依存的となったことは、阻害剤が水の代わりに亜鉛に結合したことを意味している。その時のHis64に注目すると、アセタゾラミドの場合、図４に示すようにKTは0.74となり酵素単独と比べて若干下がった。図６に示したように、経験則によるとこの値は部分的な水素結合形成を示唆しており、これらを図８の機構に当てはめると、His64と亜鉛結合水の間の水素結合形成が崩壊したことによりKTが減少し、His64とGlu106の間の水素結合において結合が繋がったり切れたりしているものと考えられる。
一方、フロセミドの場合、結晶構造解析においてフロセミドのカルボン酸とHis64 Nε2が水素結合を形成する様子が報告されており、図６に示した経験則に基づくとHis64のKTは酸性基と相互作用する値となることが予想される。そして、図４に示すようにKTの実測値は0.20であり、図６に示した経験則に基づいて導き出したKTと水素結合相互作用との規則性の正しさが証明された。
すなわち、図９にも示すように、阻害剤を添加していない状態、すなわち酵素単独の状態から阻害剤としてアセタゾラミドを添加した状態ではKTは1.0から0.74へと僅かにしか小さくならない。これは、アセタゾラミドはその構造中にスルフォンアミド基のみが存在しており、カルボキシル基のような酸性基が存在していないため、このスルフォンアミド基とCA中の亜鉛とが結合することで両者間の水素結合は崩壊したものの、His64とGlu106の間の水素結合は依然として残っているためである。
[3. Structures / behaviors reflected by signals that move due to inhibition (inhibition mechanism)]
The addition of the inhibitor made the behavior of zinc-bound histidine pH independent, meaning that the inhibitor was bound to zinc instead of water. Looking at His64 at that time, in the case of acetazolamide, as shown in FIG. 4, KT was 0.74, which was slightly lower than that of the enzyme alone. As shown in FIG. 6, this value suggests partial hydrogen bond formation according to empirical rules. When these values are applied to the mechanism shown in FIG. 8, hydrogen bond formation between His64 and zinc-bound water is disrupted. and K T is reduced by the believed that or cut lead is coupled in the hydrogen bonding between the His64 and Glu106.
On the other hand, in the case of furosemide, carboxylic acid and His64 N .epsilon.2 of furosemide in the crystal structure analysis it has been reported state of forming a hydrogen bond, K T is interacting with the acidic groups of His64 Based on empirical rule shown in FIG. 6 Expected to be a working value. As shown in FIG. 4, the measured value of K T is 0.20, and the correctness of the regularity between K T and the hydrogen bond interaction derived based on the empirical rule shown in FIG. 6 is proved.
That is, as shown in FIG. 9, KT decreases only slightly from 1.0 to 0.74 in the state where no inhibitor is added, that is, in the state where acetazolamide is added as an inhibitor from the state of the enzyme alone. This is because acetazolamide has only a sulfonamide group in its structure, and no acidic group such as a carboxyl group. Therefore, this sulfonamide group and zinc in CA bind to each other. This is because the hydrogen bond between His64 and Glu106 still remains.
一方、酵素単独の状態から阻害剤としてフロセミドを添加した状態ではKTは1.0から0.20へと大幅に小さくなった。これは、フロセミドはその構造中にスルフォンアミド基とカルボキシル基の両者が存在しており、スルフォンアミド基がCA中の亜鉛と結合することで両者間の水素結合が崩壊し、更にカルボキシル基がCA中のHis64と結合することで両者間の水素結合が崩壊したため、フロセミドと結合した状態のCA中に含まれる水素結合が大幅に減少した結果がKTの大幅減少に表れたと判断できる。KTの大幅減少は2,4-Dichloro-5-sulfamoylbenzoic acid にも見られる。
以上から、スルフォンアミド基とカルボキシル基の位置がフロセミドと同様の位置に存在する点や、CAと阻害剤との結合時に酵素単独の状態に比べてKTが小さくなる点を、高い炭酸脱水酵素阻害能を持つ被験物質のスクリーニングを行う際の指標として用いることができることが分かる。酵素単独の状態に比べてKTが大きくなる場合もまた同様と考えられる。
なお、フロセミドのアミノメチルフラン基を塩素に置換すると、亜鉛結合とHis64結合を表すｐH依存性とKTは変化しないが、酵素に対する親和性がフロセミドより10倍程度高くなりアセタゾラミドと同等になる。この阻害剤が一般に使用されない理由は、フロセミドより疎水度が低いため、灌流・血流が顕著な腎臓であっても、細胞・組織に移行しないとの理由によると考えられる。したがって、当該部位の修飾により薬物分布を調節することが可能となる。
On the other hand, KT decreased significantly from 1.0 to 0.20 when the enzyme alone was used and furosemide was added as an inhibitor. This is because furosemide has both a sulfonamide group and a carboxyl group in its structure. When the sulfonamide group is bonded to zinc in CA, the hydrogen bond between the two is broken, and the carboxyl group is also CA. It can be judged that the result of the drastic reduction of hydrogen bonds contained in CA in the state of binding to furosemide appeared to be a significant decrease in KT because the hydrogen bond between the two collapsed by binding to His64 in the inside. Significant reduction in K T is also found in 2,4-Dichloro-5-sulfamoylbenzoic acid .
Based on the above, the high carbonic anhydrase is that the position of the sulfonamide group and the carboxyl group are in the same position as furosemide, and that KT is smaller than that of the enzyme alone when binding CA and the inhibitor. It can be seen that it can be used as an index when screening a test substance having inhibitory ability. This is also considered to be the case when KT is larger than that of the enzyme alone.
Incidentally, when replacing the aminomethyl furan group of furosemide chlorine, the pH dependence and K T representing the zinc binding and His64 bond does not change, affinity is equivalent to the acetazolamide becomes 10 times higher than furosemide for the enzyme. The reason why this inhibitor is not generally used is thought to be because it has a lower hydrophobicity than furosemide, and therefore it does not migrate to cells / tissues even in the kidney where perfusion / blood flow is remarkable. Therefore, the drug distribution can be adjusted by modifying the site.
［4.これまでに報告されたCAを阻害する薬物の普遍的な相互作用原理］
フロセミドやアセタゾラミドとCAの間の相互作用に係るNMR構造情報を基に、それら低分子の化学構造式と臨床登録されているすべての阻害剤(ループ利尿薬・チアジド系利尿薬・チアジド類似利尿薬・ベンザミド)を照らし合わせて比較した。
すると図１０中に丸印で囲んだ箇所に示すように、そのすべてに亜鉛と結合するスルフォンアミド基が存在することが分かった。また、His64と相互作用するための酸性基または水素結合ドナーが存在するフロセミドタイプの阻害剤が8割で、存在しないアセタゾラミドタイプの阻害剤が残り2割であることがわかった(図１０中の●印は図１１の結晶構造と一致することを確認済み)。
[4. The universal interaction principle of drugs that have been reported to inhibit CA]
Based on NMR structural information on the interaction between furosemide or acetazolamide and CA, the chemical structural formulas of these low molecules and all clinically registered inhibitors (loop diuretics, thiazide diuretics, thiazide-like diuretics) -Benzamide) was compared and compared.
Then, as shown by the circled portions in FIG. 10, it was found that all of them have sulfonamide groups that bind to zinc. In addition, it was found that 80% of the furosemide type inhibitors in which an acidic group or hydrogen bond donor for interacting with His64 is present, and the remaining 20% of the non-existing acetazolamide type inhibitors (in FIG. 10). (The ● mark has been confirmed to match the crystal structure in FIG. 11).
これにより、図１２に示すように、I)亜鉛結合基、II)His64相互作用基、III)それらの間を連結するリンカーの疎水度(これは相互作用よりむしろ薬物の生体分布に重要と考えられる)、IV)相互作用の方向性を決定する基というように阻害剤における各官能基の役割が明確になった。
これらのI)からIV)を分子フラグメントと捉え、図１３に示すように、薬物フラグメントライブラリーからその役割に適合するフラグメントを選択し、その分子フラグメントを置換した物質を化学合成し、上記1.の1)の手法によって酵素単独時のHis64のKTを測定して水素結合相互作用を確認する。
例えば、I)亜鉛結合基の候補としては、低分子アニオンとしてmM桁の阻害が知られるCl−, Br−, I−,NO3 −,SO4 2−, NCO−, SCN−等を置換の第一対象とすることが可能である。
Thus, as shown in FIG. 12, I) zinc binding group, II) His64 interaction group, III) the hydrophobicity of the linker connecting them (this is considered to be important for drug biodistribution rather than interaction) IV), the role of each functional group in the inhibitor became clear, such as the group that determines the direction of interaction.
These I) to IV) are regarded as molecular fragments, and as shown in FIG. 13, a fragment that matches its role is selected from a drug fragment library, and a substance in which the molecular fragment is substituted is chemically synthesized, and the above 1. to confirm the hydrogen bond interactions by measuring the His64 of K T upon enzyme alone by a technique 1).
For example, as the candidate of I) a zinc binding group, Cl to mM digit inhibition is known as low-molecular anionic -, Br -, I -, NO 3 -, SO 4 2-, NCO -, SCN - , etc. The substituted It can be the first target.
また、I)亜鉛結合基とII)His64相互作用基の間の距離が重要と考えられるため、設計した低分子化合物については先に量子化学(QM)計算手法を用いた構造最適化計算手法を適用して、コンピュータシミュレーション上で予めその距離を見積もることによって実際に合成する前にある程度の絞り込みが可能と考えられる(側鎖の回転も考慮)。
発明者はこれまでにGaussian09搭載NECSX-9を用いた密度汎関数(DFT)計算による酵素の挙動解析系を確立しており、この装置を用いてその距離の見積もりを行うことが可能である。
In addition, since the distance between I) zinc binding group and II) His64 interaction group is considered important, the structural optimization calculation method using the quantum chemistry (QM) calculation method is first applied to the designed low molecular weight compound. By applying this and estimating the distance in advance on a computer simulation, it is considered possible to narrow down to a certain extent before actually synthesizing (considering rotation of the side chain).
The inventor has established an enzyme behavior analysis system by density functional (DFT) calculation using NECCSX-9 mounted on Gaussian09, and it is possible to estimate the distance using this apparatus.
以上のように、環境の酸性度と水素結合相互作用の変化を示すpKaとKTの数値の変化を追うことによって新たな阻害剤を探索することができること（図１４参照）、ここでは金属配位に係るヒスチジンのNMRスペクトルのpH依存性と、His64の酸解離定数pKa及び互変異性平衡定数KTを指標にしてCA阻害剤のスクリーニングを行うことができることが具体的に見出された。
なお、NMR測定に必要な15N標識CAは、15N塩化アンモニウムを含む培地を用いたT7系大腸菌組換え発現システムとカルボジイミド(WSC)を介して酵素阻害剤を結合させた樹脂を用いたアフィニティークロマトグラフィーによって行う系を確立しており、これにより1L培養あたり50〜100mgの精製標品を得ることが可能である。実際の測定・解析は、Bruker Avance III 500MHzNMR装置を使用し、すでにLinux（登録商標）OSマシンに搭載したNMRPipeソフトウェアを用いてシグナル解析を行い(図１５)、Windows（登録商標）OSマシン搭載KareidaGraph、Exellソフトウェアによって定数等の算出を行った。これらを使って、1サンプルを数時間でラフ解析する手法及び5日間で詳細解析する手法を確立している。
As described above, to be able to explore new inhibitors by following the change in the value of the pK a and K T showing the variation of acidity and hydrogen bonding interactions environment (see FIG. 14), wherein the metal is and pH dependence of the NMR spectra of histidine according to coordination, it is found specifically that can be screened CA inhibitors using as an index the acid dissociation constant pK a and tautomeric equilibrium constant K T of His64 It was.
The 15 N-labeled CA required for NMR measurement is an affinity using a T7 E. coli recombinant expression system using a medium containing 15 N ammonium chloride and a resin in which an enzyme inhibitor is bound via carbodiimide (WSC). A system for performing chromatography is established, whereby it is possible to obtain a purified preparation of 50 to 100 mg per 1 L culture. For actual measurement and analysis, a Bruker Avance III 500MHz NMR device was used, and signal analysis was performed using NMRPipe software already installed on a Linux (registered trademark) OS machine (Fig. 15). KareidaGraph installed on a Windows (registered trademark) OS machine The constants were calculated by Exell software. Using these, we have established a method for rough analysis of one sample in a few hours and a method for detailed analysis in 5 days.
[発明の将来展望]
ヒスチジンは、古来より生理的pH付近においてpKaをもつ唯一のアミノ酸側鎖として注目されており、酸塩基触媒や金属配位といった蛋白質の機能発現に重要な役割を担うことが知られている。
炭酸脱水酵素(CA)のように活性部位ヒスチジンがそのような発現に関与する場合、ヒスチジンシグナルを効果的な創薬プローブとして適切にモニターするならば、相互作用を検出するための手段として非常に有用であり、将来、多くの蛋白質に適用される可能性が極めて高いと考えられる。
[Future perspective of the invention]
Histidine has attracted attention since ancient times as the only amino acid side chain having a pKa near physiological pH, and is known to play an important role in the expression of protein functions such as acid-base catalysis and metal coordination.
If the active site histidine is involved in such expression, such as carbonic anhydrase (CA), then the histidine signal can be a great tool for detecting interactions if it is properly monitored as an effective drug discovery probe. It is useful and is very likely to be applied to many proteins in the future.
発明者は既に遺伝子クローニング(mRNA抽出型・遺伝子合成型を含む)・発現系構築技術を有しており、かつバイオインフォマティクス技術をヒスチジンが機能する蛋白質探索に適用し、そこで確立された手法を他の蛋白質に適用することが可能である（図１６参照）。
ヒスチジンを用いてこのようなスクリーニング手法が可能であることを示すならば、他のアミノ酸もまた同様に開発されることが可能であり、最終的にアミノ酸側鎖網羅型のスクリーニング手法が視野に入るため、その波及効果は絶大である。
The inventor already has gene cloning (including mRNA extraction type and gene synthesis type) and expression system construction technology, and applied bioinformatics technology to protein search for histidine function, and other methods established there (See FIG. 16).
If histidine is used to show that such a screening technique is possible, other amino acids can be developed in the same way, and finally an amino acid side chain comprehensive screening technique is in the field of view. Therefore, the ripple effect is tremendous.
一方、本研究で解明されるCAの触媒機構からその触媒必須因子（亜鉛結合水、His64、Glu106）を抽出、かつそれらの官能基を人工的に適宜配置することによって、酵素を模倣した二酸化炭素水和触媒を開発することが可能である（図１７参照）。その機能的な配置は、ヒスチジンの挙動によって検出・確認することが可能であるため、ここでもまた発明者が見出した経験則を基盤とする「Histidine-based NMRスクリーニング法」によって探索することが可能である。
本研究をこのような触媒低分子化の方向に進めるならば、二酸化炭素水和技術によって、植物が光合成前段階で行う大気中二酸化炭素の取込みを模倣するバイオミメティックスがさらに現実味を帯び、環境分野の課題解決に大きくアプローチすることが可能である。
このように、相互作用の解明にかかる真の構造生命科学によるスクリーニング技術を基盤とすることで、合理的創薬と二酸化炭素回収という2つの大きなライフサイエンス課題を解決できる。
On the other hand, carbon dioxide that imitates an enzyme by extracting the essential factors of the catalyst (zinc-bound water, His64, Glu106) from the CA catalytic mechanism elucidated in this study and artificially arranging these functional groups appropriately It is possible to develop a hydration catalyst (see FIG. 17). Since its functional arrangement can be detected and confirmed by the behavior of histidine, it can be searched here again by the “Histidine-based NMR screening method” based on the empirical rule found by the inventor. It is.
If this research is advanced in the direction of lowering the molecular weight of the catalyst, biomimetics that mimic the uptake of atmospheric carbon dioxide by plants in the pre-photosynthesis stage will become even more realistic with the carbon dioxide hydration technology. It is possible to approach the problem solution greatly.
In this way, the two major life science issues of rational drug discovery and carbon dioxide recovery can be solved by using the screening technology based on the true structural life science to clarify the interaction.
本発明は、創薬効果的プローブとして機能性アミノ酸ヒスチジンの挙動を利用する炭酸脱水酵素阻害剤のNMRスクリーニング法(Histidine-based NMRスクリーニング法)に関するものであり、産業上利用可能である。
The present invention relates to a carbonic anhydrase inhibitor NMR screening method (Histidine-based NMR screening method) that utilizes the behavior of a functional amino acid histidine as a drug discovery effective probe, and is industrially applicable.
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