GRGDsP acts as inhibitor of cell attachment to fibronectin but not to vitronectin.

RGD肽-說明

RGD肽是指含有由Arg-Gly-Asp三個氨基酸組成的序列多肽，有直線肽和環(huán)肽之分。它們是許多細胞外基質(zhì)蛋白（如VN、FN、FGN、膠原等）等最小識別短肽序列。

研究發(fā)現(xiàn)，RGD序列肽具有廣泛的生物活性，可用于心血管疾病、骨質(zhì)疏松和炎癥等疾病的治療，還可以預(yù)防和治療由細胞粘附異常而導(dǎo)致的腫瘤，尤其是發(fā)展性腫瘤的轉(zhuǎn)移；另一方面，RGD 序列肽又可作為興奮劑，促進損傷的器官與組織的再生、傷口的愈合等等，RGD作為某些整合素的受體，其選擇性部分依賴于RGD的構(gòu)象以及RGD周圍的氨基酸殘基。

為此，近幾年，許多科技工作者合成了一系列RGD三肽、四肽、五肽等，還合成了RGD環(huán)肽、雙線肽、RGD模擬肽等等。為了滿足客戶對各種RGD序列肽的需求，專肽生物提供最廣泛的RGD序列肽庫，以滿足科研工作者對RGD肽的需求。

專肽生物提供各種RGD肽的現(xiàn)貨，縮短科研工作者的項目時間，例如c(RGDfK)、c(RGDfC)、c(RADyK)、c(RGDyK)、c(RADfC)、環(huán)狀多肽c(RGDfK)-巰基乙酸、c(RGDfK)-PEG2-巰基乙酸、Mpa-Ahx-c(RGDfK)、環(huán)狀多肽c(RGDfK)-半胱氨酸、DOTA-c(RGDfK)、NOTA-c(RGDfK)、NOTA-c(RGDyK)、DOTA-c(RGDyK)、E[c(RGDfK)]2、E[c(RGDyK)]2、DDDDD-c(RGDfK)等等，具體可咨詢銷售人員。

很多蛋白在細胞中非常容易被降解，或被標(biāo)記，進而被選擇性地破壞。但含有部分D型氨基酸的多肽則顯示了很強的抵抗蛋白酶降解能力。

定義
酶是用于生化反應(yīng)的非常有效的催化劑。它們通過提供較低活化能的替代反應(yīng)途徑來加快反應(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)測新的抑制劑：合理的藥物設(shè)計使用酶活性位點的三維結(jié)構(gòu)來預(yù)測哪些分子可能是抑制劑1。已經(jīng)開發(fā)了用于識別酶抑制劑的基于計算機的方法，例如分子力學(xué)和分子對接。
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位置具有一個新的內(nèi)酰胺部分，而對胰蛋白酶選擇性最高的兩種抑制劑在P1位置具有一個與S1特異性位點結(jié)合的胍基哌啶基。凝血酶的抑制動力學(xué)從慢到快變化，而對于胰蛋白酶，抑制的動力學(xué)在所有情況下都快。根據(jù)兩步機理2中穩(wěn)定過渡態(tài)絡(luò)合物的緩慢形成來檢驗動力學(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，活性位點通過與底物的相互作用不斷重塑。
在眾多生物活性肽的成熟過程中，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)揭示了類似于兩個鋅外肽酶的α/β支架，但有多個插入和缺失，特別是在活性位點區(qū)域。幾種活性位點突變酶的結(jié)構(gòu)分析為針對QC相關(guān)疾病5的抑制劑的合理設(shè)計提供了結(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)合可以阻止底物進入酶的活性位點和/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-競爭性和非競爭性?？赡嬉种苿?nbsp;以非共價相互作用（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 對單一酶發(fā)揮作用。大多數(shù)毒藥通過特異性抑制酶發(fā)揮作用。競爭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與酶相互作用，但是沒有反應(yīng)發(fā)生。非競爭性抑制劑是與酶相互作用但通常不在活性位點相互作用的物質(zhì)。非競爭性抑制劑的凈作用是改變酶的形狀，從而改變活性位點，從而使底物不再能與酶相互作用而產(chǎn)生反應(yīng)。非競爭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鍵。這些抑制劑可以在活性位點附近或附近起作用。
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)的一部分進行設(shè)計和生產(chǎn)6。
天然毒物 通常是酶抑制劑，已進化為保護植物或動物免受天敵的侵害。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。
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.

多肽H2N-Gly-Arg-Gly-Asp-DSer-Pro-COOH的合成步驟：

1、合成CTC樹脂：稱取1.98g CTC Resin（如初始取代度約為1.01mmol/g）和2.4mmol Fmoc-Pro-OH于反應(yīng)器中，加入適量DCM溶解氨基酸（需要注意，此時CTC樹脂體積會增大好幾倍，避免DCM溶液過少），再加入6.0mmol DIPEA（Mw：129.1,d：0.740g/ml），反應(yīng)2-3小時后，可不抽濾溶液，直接加入1ml的HPLC級甲醇，封端半小時。依次用DMF洗滌2次，甲醇洗滌1次，DCM洗滌一次，甲醇洗滌一次，DCM洗滌一次，DMF洗滌2次（這里使用甲醇和DCM交替洗滌，是為了更好地去除其他溶質(zhì)，有利于后續(xù)反應(yīng)）。得到  Fmoc-Pro-CTC Resin。結(jié)構(gòu)圖如下：

2、脫Fmoc：加3倍樹脂體積的20%Pip/DMF溶液，鼓氮氣30分鐘，然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Pro-CTC Resin 。（此步驟脫除Fmoc基團，茚三酮檢測為藍色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取6.0mmol Fmoc-DSer(tBu)-OH 氨基酸，加入到上述樹脂里，加適當(dāng)DMF溶解氨基酸，再依次加入12.0mmol DIPEA，5.7mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測為無色。用2倍樹脂體積的DMF 洗滌3次樹脂。（洗滌樹脂，去掉殘留溶劑，為下一步反應(yīng)做準(zhǔn)備）。得到Fmoc-DSer(tBu)-Pro-CTC Resin。氨基酸：DIPEA：HBTU：樹脂=3：6：2.85：1（摩爾比）。結(jié)構(gòu)圖如下：

4、依次循環(huán)步驟二、步驟三，依次得到

H2N-DSer(tBu)-Pro-CTC Resin

Fmoc-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

H2N-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

Fmoc-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

H2N-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

Fmoc-Arg(Pbf)-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

H2N-Arg(Pbf)-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

Fmoc-Gly-Arg(Pbf)-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin

以上中間結(jié)構(gòu)，均可在專肽生物多肽計算器-多肽結(jié)構(gòu)計算器中，一鍵畫出。

最后再經(jīng)過步驟二得到 H2N-Gly-Arg(Pbf)-Gly-Asp(OtBu)-DSer(tBu)-Pro-CTC Resin，結(jié)構(gòu)如下：

5、切割：6倍樹脂體積的切割液（或每1g樹脂加8ml左右的切割液），搖床搖晃 2小時，過濾掉樹脂，用冰無水乙醚沉淀濾液，并用冰無水乙醚洗滌沉淀物3次，最后將沉淀物放真空干燥釜中，常溫干燥24小試，得到粗品H2N-Gly-Arg-Gly-Asp-DSer-Pro-COOH。結(jié)構(gòu)圖見產(chǎn)品結(jié)構(gòu)圖。

切割液選擇：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前兩種適合沒有容易氧化的氨基酸，例如Trp、Cys、Met。第三種適合幾乎所有的序列。）

6、純化凍干：使用液相色譜純化，收集目標(biāo)峰液體，進行凍干，獲得蓬松的粉末狀固體多肽。不過這時要取小樣復(fù)測下純度 是否目標(biāo)純度。

7、最后總結(jié)：

杭州專肽生物技術(shù)有限公司（ALLPEPTIDE http://amynixphotography.com）主營定制多肽合成業(yè)務(wù)，提供各類長肽，短肽，環(huán)肽，提供各類修飾肽，如：熒光標(biāo)記修飾（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基團修飾肽（疊氮、炔基、DBCO、DOTA、NOTA等），同位素標(biāo)記肽（N15、C13），訂書肽（Stapled Peptide）,脂肪酸修飾肽（Pal、Myr、Ste），磷酸化修飾肽（P-Ser、P-Thr、P-Tyr），環(huán)肽（酰胺鍵環(huán)肽、一對或者多對二硫鍵環(huán)），生物素標(biāo)記肽，PEG修飾肽，甲基化修飾肽

以上所有內(nèi)容，為專肽生物原創(chuàng)內(nèi)容，請勿發(fā)布到其他網(wǎng)站上。