Ac-DEVD-CMK是一種細(xì)胞可滲透的、不可逆的胱天蛋白酶-3以及胱天蛋白酶-6、-7、-8和-10的抑制劑。它通常在高達(dá)100μM的濃度下用于檢測(cè)胱天蛋白酶-3依賴性細(xì)胞凋亡在生物系統(tǒng)中的作用。

Ac-DEVD-CMK is a cell-permeable, and irreversible inhibitor of caspase-3 as well as caspase-6, -7, -8, and -10. It is commonly used at concentrations up to 100 μM to examine the role of caspase-3-dependent apoptosis in biological systems.

定義
酶是用于生化反應(yīng)的非常有效的催化劑。它們通過(guò)提供較低活化能的替代反應(yīng)途徑來(lái)加快反應(yīng)速度。酶作用于底物并產(chǎn)生產(chǎn)物。一些物質(zhì)降低或什至停止酶的催化活性被稱為抑制劑。
發(fā)現(xiàn)
1965年，Umezawa H分析了微生物產(chǎn)生的酶抑制劑，并分離出了抑制亮肽素和抗痛藥的胰蛋白酶和木瓜蛋白酶，乳糜蛋白酶抑制的胰凝乳蛋白酶，胃蛋白酶抑制素抑制胃蛋白酶，泛磷酰胺抑制唾液酸酶，烏藤酮抑制酪氨酸羥化酶，多巴汀抑制多巴胺3-羥硫基嘧啶和多巴胺3-羥色胺酶酪氨酸羥化酶和多巴胺J3-羥化酶。最近，一種替代方法已應(yīng)用于預(yù)測(cè)新的抑制劑：合理的藥物設(shè)計(jì)使用酶活性位點(diǎn)的三維結(jié)構(gòu)來(lái)預(yù)測(cè)哪些分子可能是抑制劑1。已經(jīng)開發(fā)了用于識(shí)別酶抑制劑的基于計(jì)算機(jī)的方法，例如分子力學(xué)和分子對(duì)接。
結(jié)構(gòu)特征
已經(jīng)確定了許多抑制劑的晶體結(jié)構(gòu)。已經(jīng)確定了三種與凝血酶復(fù)合的高效且選擇性的低分子量剛性肽醛醛抑制劑的晶體結(jié)構(gòu)。這三種抑制劑全部在P3位置具有一個(gè)新的內(nèi)酰胺部分，而對(duì)胰蛋白酶選擇性最高的兩種抑制劑在P1位置具有一個(gè)與S1特異性位點(diǎn)結(jié)合的胍基哌啶基。凝血酶的抑制動(dòng)力學(xué)從慢到快變化，而對(duì)于胰蛋白酶，抑制的動(dòng)力學(xué)在所有情況下都快。根據(jù)兩步機(jī)理2中穩(wěn)定過(guò)渡態(tài)絡(luò)合物的緩慢形成來(lái)檢驗(yàn)動(dòng)力學(xué)。
埃米爾•菲舍爾（Emil Fischer）在1894年提出，酶和底物都具有特定的互補(bǔ)幾何形狀，彼此恰好契合。這稱為“鎖和鑰匙”模型3。丹尼爾·科什蘭（Daniel Koshland）提出了誘導(dǎo)擬合模型，其中底物和酶是相當(dāng)靈活的結(jié)構(gòu)，當(dāng)?shù)孜锱c酶4相互作用時(shí)，活性位點(diǎn)通過(guò)與底物的相互作用不斷重塑。
在眾多生物活性肽的成熟過(guò)程中，需要由其谷氨酰胺（或谷氨酰胺）前體形成N末端焦谷氨酸（pGlu）。游離形式并與底物和三種咪唑衍生抑制劑結(jié)合的人QC的結(jié)構(gòu)揭示了類似于兩個(gè)鋅外肽酶的α/β支架，但有多個(gè)插入和缺失，特別是在活性位點(diǎn)區(qū)域。幾種活性位點(diǎn)突變酶的結(jié)構(gòu)分析為針對(duì)QC相關(guān)疾病5的抑制劑的合理設(shè)計(jì)提供了結(jié)構(gòu)基礎(chǔ)。
作用方式
酶是催化化學(xué)反應(yīng)的蛋白質(zhì)。酶與底物相互作用并將其轉(zhuǎn)化為產(chǎn)物。抑制劑的結(jié)合可以阻止底物進(jìn)入酶的活性位點(diǎn)和/或阻止酶催化其反應(yīng)。抑制劑的種類繁多，包括：非特異性，不可逆，可逆-競(jìng)爭(zhēng)性和非競(jìng)爭(zhēng)性。可逆抑制劑 以非共價(jià)相互作用（例如疏水相互作用，氫鍵和離子鍵）與酶結(jié)合。非特異性抑制方法包括最終使酶的蛋白質(zhì)部分變性并因此不可逆的任何物理或化學(xué)變化。特定抑制劑 對(duì)單一酶發(fā)揮作用。大多數(shù)毒藥通過(guò)特異性抑制酶發(fā)揮作用。競(jìng)爭(zhēng)性抑制劑是任何與底物的化學(xué)結(jié)構(gòu)和分子幾何結(jié)構(gòu)非常相似的化合物。抑制劑可以在活性位點(diǎn)與酶相互作用，但是沒有反應(yīng)發(fā)生。非競(jìng)爭(zhēng)性抑制劑是與酶相互作用但通常不在活性位點(diǎn)相互作用的物質(zhì)。非競(jìng)爭(zhēng)性抑制劑的凈作用是改變酶的形狀，從而改變活性位點(diǎn)，從而使底物不再能與酶相互作用而產(chǎn)生反應(yīng)。非競(jìng)爭(zhēng)性抑制劑通常是可逆的。不可逆抑制劑與酶形成牢固的共價(jià)鍵。這些抑制劑可以在活性位點(diǎn)附近或附近起作用。
功能
工業(yè)應(yīng)用中， 酶在商業(yè)上被廣泛使用，例如在洗滌劑，食品和釀造工業(yè)中。蛋白酶用于“生物”洗衣粉中，以加速蛋白質(zhì)在諸如血液和雞蛋等污漬中的分解。商業(yè)上使用酶的問(wèn)題包括：它們是水溶性的，這使得它們難以回收，并且一些產(chǎn)物可以抑制酶的活性（反饋抑制）。
藥物分子，許多藥物分子都是酶抑制劑，藥用酶抑制劑通常以其特異性和效力為特征。高度的特異性和效力表明該藥物具有較少的副作用和較低的毒性。酶抑制劑在自然界中發(fā)現(xiàn)，并且也作為藥理學(xué)和生物化學(xué)的一部分進(jìn)行設(shè)計(jì)和生產(chǎn)6。
天然毒物 通常是酶抑制劑，已進(jìn)化為保護(hù)植物或動(dòng)物免受天敵的侵害。這些天然毒素包括一些已知最劇毒的化合物。
神經(jīng)氣體（ 例如二異丙基氟磷酸酯（DFP））通過(guò)與絲氨酸的羥基反應(yīng)生成酯，從而抑制了乙酰膽堿酯酶的活性位點(diǎn)。
參考
1、Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.
2、Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.
3、Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.
4、Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.
5、Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.
6、Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

Definition
Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction pathway of lower activation energy. Enzyme acts on substrate and gives rise to a product. Some substances reduce or even stop the catalytic activities of enzymes are called inhibitors.

Discovery
In 1965, Umezawa H analysed enzyme inhibitors produced by microorganisms and isolated leupeptin and antipain inhibiting trypsin and papain, chymostatin inhibiting chymotrypsin, pepstatin inhibiting pepsin, panosialin inhibiting sialidases, oudenone inhibiting tyrosine hydroxylase, dopastin inhibiting dopamine 3-hydroxylase, aquayamycin and chrothiomycin inhibiting tyrosine hydroxylase and dopamine J3-hydroxylase . Recently, an alternative approach has been applied to predict new inhibitors: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors 1. Computer-based methods for identifying inhibitor for an enzyme have been developed, such as molecular mechanics and molecular docking.

Structural Characteristics
The crystal structures of many inhibitors have been determined. The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined. All the three inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism 2.

Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model 3. Daniel Koshland suggested induced fit model where substrate and enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 4.

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The structural analyses of several active-site-mutant enzymes provide a structural basis for the rational design of inhibitors against QC-associated disorders 5.

Mode of Action
Enzymes are proteins that catalyze chemical reactions. Enzymes interact with substrate and convert them into products. Inhibitor binding can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. Reversible inhibitors bind to enzymes with non-covalent interactions like hydrophobic interactions, hydrogen bonds, and ionic bonds. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible. Specific Inhibitors exert their effects upon a single enzyme. Most poisons work by specific inhibition of enzymes. A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site.  The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible. Irreversible Inhibitors form strong covalent bonds with an enzyme.  These inhibitors may act at, near, or remote from the active site .

Functions
Industrial application, enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in 'biological' washing powders to speed up the breakdown of proteins in stains like blood and egg. Problems using enzymes commercially include: they are water soluble which makes them hard to recover and some products can inhibit the enzyme activity (feedback inhibition) .

Drug molecules, many drug molecules are enzyme inhibitors and a medicinal enzyme inhibitor is usually characterized by its specificity and its potency. A high specificity and potency suggests that a drug will have fewer side effects and less toxic. Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry 6.

Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known.

Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.

References

Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.

Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.

Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.

Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.

Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

定義
細(xì)胞凋亡或程序性細(xì)胞死亡是多細(xì)胞生物發(fā)育和健康的正常組成部分。細(xì)胞響應(yīng)各種刺激而死亡，在細(xì)胞凋亡期間，它們以受控，受控的方式死亡。

發(fā)現(xiàn)
1885年， Flemming W描述了程序性細(xì)胞死亡的過(guò)程。約翰·克爾（John Kerr）在1960年代后期的發(fā)現(xiàn)最初被稱為“收縮壞死”，但后來(lái)改名為“細(xì)胞凋亡”，是在他對(duì)大鼠急性肝損傷的研究中，他的注意力被好奇的肝細(xì)胞死亡形式引起的 ^1,2。  1972年，Kerr提出了術(shù)語(yǔ)“細(xì)胞凋亡”的意思是控制細(xì)胞缺失的機(jī)制，它似乎在調(diào)節(jié)動(dòng)物細(xì)胞群中與有絲分裂起著互補(bǔ)但相反的作用。它的形態(tài)學(xué)特征表明它是一種活躍的，固有編程的現(xiàn)象，并且已經(jīng)表明它可以被多種環(huán)境刺激所引發(fā)或抑制， ³。 

結(jié)構(gòu)特征
Bcl-2家族成員之間的異二聚化是調(diào)節(jié)程序性細(xì)胞死亡的關(guān)鍵事件。通過(guò)確定存活蛋白Bcl-xL和Bcl-2相關(guān)蛋白Bak的促死亡區(qū)域之間的復(fù)合物的溶液結(jié)構(gòu)，研究了異二聚體形成的分子基礎(chǔ)。突變型Bak肽的結(jié)構(gòu)和結(jié)合親和力表明Bak肽采用兩親性螺旋，通過(guò)疏水和靜電相互作用與Bcl-xL相互作用。全長(zhǎng)Bak的突變會(huì)破壞任一類型的相互作用，從而抑制Bak與Bcl-xL ⁴異源二聚的能力。

通過(guò)核磁共振波譜（NMR）確定與Bcl-xL具有生物活性的缺失突變體復(fù)合的16-氨基酸肽的結(jié)構(gòu)。由總共2813個(gè)NMR約束確定結(jié)構(gòu)，并通過(guò)NMR數(shù)據(jù)很好地定義了結(jié)構(gòu)。當(dāng)與Bcl-xL復(fù)合時(shí)，Bak肽形成螺旋。Bak肽的COOH末端部分主要與BH2和BH3區(qū)的殘基相互作用。黑色素瘤細(xì)胞凋亡抑制劑（ML-IAP）是一種有效的抗凋亡蛋白，在許多黑色素瘤細(xì)胞系中上調(diào)，但在大多數(shù)正常成人組織中均未表達(dá)。在人類癌癥中，IAP蛋白（例如ML-IAP或普遍表達(dá)的X染色體連接的IAP（XIAP））的過(guò)表達(dá)已顯示可抑制多種刺激誘導(dǎo)的細(xì)胞凋亡。⁵。

作用方式
一旦收到指示細(xì)胞進(jìn)行凋亡的特定信號(hào)，細(xì)胞中就會(huì)發(fā)生許多明顯的變化。稱為胱天蛋白酶的蛋白質(zhì)家族通常在凋亡的早期被激活。這些蛋白質(zhì)分解或切割正常細(xì)胞功能所需的關(guān)鍵細(xì)胞成分，包括細(xì)胞骨架中的結(jié)構(gòu)蛋白和核蛋白（例如DNA修復(fù)酶）。半胱天冬酶還可以活化其他降解酶，例如DNase，其開始切割細(xì)胞核中的DNA。

凋亡細(xì)胞在凋亡過(guò)程中表現(xiàn)出獨(dú)特的形態(tài)。通常，在細(xì)胞骨架中的lamins和肌動(dòng)蛋白絲分裂后，細(xì)胞開始收縮。染色質(zhì)在細(xì)胞核中的分解通常會(huì)導(dǎo)致核濃縮，并且在許多情況下，凋亡細(xì)胞的細(xì)胞核呈“馬蹄形”的外觀。細(xì)胞繼續(xù)收縮，將自身包裝成可被巨噬細(xì)胞去除的形式。有許多機(jī)制可以通過(guò)誘導(dǎo)細(xì)胞凋亡。細(xì)胞對(duì)這些刺激中任何一種的敏感性可能會(huì)因多種因素而異，例如促凋亡和抗凋亡蛋白（例如Bcl-2蛋白或凋亡蛋白的抑制劑）的表達(dá)，刺激的嚴(yán)重程度和細(xì)胞周期的階段。Bcl-2蛋白家族在調(diào)節(jié)多種刺激誘導(dǎo)的凋亡細(xì)胞死亡中起著核心作用。該家族中的某些蛋白質(zhì)，包括Bcl-2和Bcl-xL，可以抑制程序性細(xì)胞死亡，而其他蛋白質(zhì)，例如Bax和Bak，可以促進(jìn)細(xì)胞凋亡 ^6、7。

功能

對(duì)于發(fā)育，細(xì)胞凋亡與有絲分裂一樣是正常發(fā)育所必需的。例子：tail變尾時(shí)into的吸收被青蛙吸收。

生物體的完整性需要凋亡來(lái)破壞對(duì)生物體完整性構(gòu)成威脅的細(xì)胞。例子：感染了病毒的細(xì)胞⁸。

免疫系統(tǒng)的細(xì)胞，一種細(xì)胞介導(dǎo)的免疫反應(yīng)減弱，必須去除效應(yīng)細(xì)胞以防止它們攻擊機(jī)體成分。CTLs互相誘導(dǎo)凋亡，甚至自身誘導(dǎo)凋亡⁹。

具有DNA損傷，破壞其基因組的細(xì)胞會(huì)導(dǎo)致細(xì)胞破壞正常的胚胎發(fā)育，導(dǎo)致先天缺陷變成癌。

參考

1.     Kerr JF (1965). A histochemical study of hypertrophy and ischaemic injury of rat liver with special reference to changes in lysosomes. Journal of Pathology and Bacteriology, 90(90):419-435.

2.     Kerr JF, Wyllie AH, Currie AR (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer., 26(4):239-257.

3.     O'Rourke MG, Ellem KA (2000). John Kerr and apoptosis. Med. J. Aust., 173(11-12): 616-617.

4.     Franklin MC, Kadkhodayan S, Ackerly H, Alexandru D, Distefano MD, Elliott LO, Flygare JA, Mausisa G, Okawa DC, Ong D, Vucic D, Deshayes K, Fairbrother WJ (2003). Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP). Biochemistry, 42(27):8223-8231.

5.     Sattler M, Liang H, Nettesheim D, Meadows RP, Harlan JE, Eberstadt M, Yoon HS, Shuker SB, Chang BS, Minn AJ, Thompson CB, Fesik SW (1997). Structure of bcl-xl-bak peptide complex: recognition between regulators of apoptosis. Science, 275(5302):983-986.

6.     Hanada M, Aimé-Sempé C, Sato T, Reed JC (1995). Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J. Biol. Chem., 270(20):11962-11969.

7.     Cheng EHY, Levine B, Boise LH, Thompson CB, Hardwic JM (1996). Bax-independent inhibition of apoptosis by Bcl-xL.Nature, 379:554-556.

8.     Alimonti JB, Ball TB, Fowke KR (2003). Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS. J Gen Virology., 84(84): 1649-1661.

9.     Werlen G, Hausmann B, Naeher D, Palmer E (2003). Signaling life and death in the thymus: timing is everything. Science. 299(5614):1859-1863.

Ac-DEVD-CMK irreversibly inhibits caspase-3. It is also active towards caspases-6, -7, -8, and -10.