合成Exendin-4的体外人血浆稳定性研究

为了解决Ⅱ型糖尿病患者注射用药频繁的问题, 需要比Exendin-4更加稳定的GLP-1的激动剂, 本文的研究目的是找到Exendin-4较易水解的氨基酸位点, 为设计新的长效的Exendin-4模拟肽提供相关信息.     

Exendin-4中13号残基的分子动力学模拟

Exendin-4作为胰腺GLP-1受体上的一种有效的激活剂, 是一种含有39个氨基酸残基的多肽, 其第13号氨基酸Gln突变为Tyr, 使活性增强. 应用分子动力学模拟方法, 分别优化了突变前后, Exendin-4与蛋白的复合物结构, 并对整体结构的性质、静电势、相互作用模式及能量进行了分析. 阐明了Gln突变为Tyr的活性增强的内在原因, 结果表明, 突变的Exendin-4能够通过改变自身结构的局部柔性调整与蛋白受体相互作用, 从而可以改善Exendin-4与其蛋白受体的结合能力.     

SDS胶束溶液中Exendin-4活性类似物的结构分析

Exendin-4（EX-4）是一种治疗II型糖尿病的潜在药物。研究发现用β-Asp和Gln同时替换EX-4中的Glu3和Tyr13得到的EX-4类似物比EX-4有更好的降糖效果和抗水解的稳定性。本文用核磁共振方法研究了SDS胶束溶液中EX-4和这种活性类似物的三维空间结构。结果表明在SDS胶束溶液中EX-4活性类似物与EX-4原始肽的结构都由具有α螺旋卷曲构象的中间区域和灵活的N端区域及形成Trp-cage结构的C端区域构成,但与原始肽结构不同的是类似物的螺旋区域向N端扩展了3个残基,螺旋结构向N端的延长可能改善了底物与受体的亲和力,提高了生物活性。     

具有新型核心结构的肽类BACE1抑制剂的设计、合成及活性评价

以KMI-008为先导化合物,4-羟基或氨基取代的脯氨酸模拟其Pns,设计并合成了19条肽序列,用时间分辨荧光法测定目标肽体外对BACE1的抑制作用. 所合成化合物对BACE1有一定的抑制作用,其中两个化合物(LK-MX-41、LK-MX-42)与先导化合物活性相近,其核心结构由L-Phe-D-Pro组成,可以模拟BACE1的催化中心与底物相互作用的过渡态;含有该结构的肽序列,有可能成为研究肽类BACE1抑制剂的另一途径. 应用Autodock 4将所合成化合物与BACE1进行对接,结合体外活性测试结果对构效关系进行了初步探讨. 先导化合物及目标肽用固相法合成,经RP-HPLC测定纯度、ESI-MS确定相对分子量.     

抗菌肽SAMP1及其类似肽的构效关系

以人工合成抗菌肽1(Synthetic antimicrobial peptide 1, SAMP1)为研究模板, 采用氨基酸序列重排、 不同的带正电荷氨基酸残基和疏水性氨基酸残基取代等方法, 设计合成了8条SAMP1类似肽. 利用生物信息学软件预测了SAMP1及其类似肽的理化性质; 采用圆二色光谱(CD)技术测定其在不同环境下二级结构的变化; 采用噻唑蓝(MTT)法测定其抗菌活性; 通过红细胞溶血实验评估了这些多肽的溶血性. 结果表明, 大部分类似肽具有较低的溶血毒性和较高的广谱抗菌活性. CD光谱分析结果显示, 大部分类似肽二级结构以α螺旋和无规则卷曲为主, 在体积分数为50%的2,2,2-三氟乙醇(TFE)溶液中, α螺旋结构比例增加. 与母肽SAMP1相比, 经序列重排后得到的SAMP1-A1, SAMP1-A2和SAMP1-A3的抗菌活性变化不大, 但序列中正电荷氨基酸残基均匀分布的类似肽SAMP1-A2的溶血毒性增加. 用精氨酸(Arg)取代SAMP1序列中的赖氨酸(Lys)得到的类似肽SAMP1-A4的抗菌活性增强, 同时溶血毒性降低. 用疏水性较强的异亮氨酸(Ile)和缬氨酸(Val)取代SAMP1中的疏水性氨基酸残基, 得到的类似肽SAMP1-A5和SAMP1-A7的抗菌活性急剧降低; 用疏水性较弱的色氨酸(Trp)取代SAMP1中的疏水性氨基酸残基, 得到的类似肽SAMP1-A8的抗菌活性增强, 同时溶血毒性提高.     

新型抗菌肽的设计、活性研究及与磷脂相互作用的计算模拟

设计合成了多个具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性, 发现其杀菌活性顺序为长链肽>环状肽>短链肽, 特别是线性的Linear-KT和Linear-KS对多种革兰氏阴性菌和阳性菌均具有较高的杀菌活性. 采用MTT法考察了Linear-KT和Linear-KS对正常细胞的毒性, 其中Linear-KS表现出较低的细胞毒性, 优于阳性对照多粘菌素B. 利用计算模拟的方法计算了多肽与细菌细胞膜中磷脂酰甘油(DMPG)的相互作用. 结果表明, 多肽和DMPG的结合能也表现出长链肽>环状肽>短链肽的规律, 特别是Linear-KT和Linear-KS具有较高的结合能. 长链肽含有2个活性序列, 可提供多个荷正电的氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 同时, 柔性的结构及Linear-KT和Linear-KS中丝氨酸和苏氨酸的β碳上的羟基可与磷脂上的羰基形成多个氢键, 进一步增大了结合能. 计算模拟的方法为抗菌肽的杀菌活性从理论上提供了一定的依据.     

海地瓜蛋白水解物中ACE抑制肽的分离纯化及合成

采用Sephadex G-25凝胶柱层析、SP Sephadex C-25 阳离子交换层析和反相高效液相色谱等方法对海地瓜水解产物进行分离纯化, 得到了一种新的强活性ACE抑制肽, 其氨基酸序列为MEGAQEAQGD, IC₅₀值为15.9 μmol/L. 采用逐步缩合和片段缩合的方法对该抑制肽进行了设计合成. 合成肽的纯度为99.72%, 分子量与序列结构均与理论值相符. 研究发现, 抑制肽与胃蛋白酶和糜蛋白酶水解反应后, 活性增强了3.5倍. 动物实验结果表明, 剂量为3 μmol/kg的抑制肽对大鼠自发性高血压具有明显的降压效果.     

胆固醇修饰的非天然HR序列肽类HIV-1融合抑制剂的合成及活性初筛

将胆固醇分子通过1个半胱氨酸侧链硫醚键和1个β-丙氨酸连接臂引入到所设计的非天然HR序列抗HIV融合活性多肽的C端和N端,合成了与天然C肽序列同源性很低的非天然序列的类肽抗HIV融合分子,以考察胆固醇修饰对非天然HR序列活性的影响,探讨克服耐药性的新思路.生物活性评价结果表明,胆固醇与HR肽C端结合物抑制HIV融合活性显著提高,而连接到N端的序列则完全失去了抗病毒活性,表明所设计的非天然序列确实具有与天然序列类似的作用机制.     

罂栗花粉中水溶性肽的分离纯化、序列测定和合成的研究

从罂栗花粉中分离得到含21, 17, 13和16个氨基酸残基的四种肽PSPP1～4, 测定了它们的序列。用固相法合成了PSPP2～4, 经HPLC和电泳法对比, 确定了分离肽的一级结构。用FT-IR、CD谱初步讨论了它们在固态和溶液中的二级结构。通过T-淋巴细胞转化实验等生理活性测定方法表明它们具有一定的促免疫活性。     

罂栗花粉中水溶性肽的分离纯化、序列测定和合成的研究

从罂栗花粉中分离得到含21, 17, 13和16个氨基酸残基的四种肽PSPP1～4, 测定了它们的序列。用固相法合成了PSPP2～4, 经HPLC和电泳法对比, 确定了分离肽的一级结构。用FT-IR、CD谱初步讨论了它们在固态和溶液中的二级结构。通过T-淋巴细胞转化实验等生理活性测定方法表明它们具有一定的促免疫活性。     

Synthesis and biological evaluation of leucine enkephalin turn mimetics

A cyclic Leu-enkephalin mimetic containing a 7-membered ring, and two linear analogues, has been prepared on solid phase. In the cyclic mimetic the intramolecular (1-4) hydrogen bond found in crystalline Leu-enkephalin has been replaced by an ethylene bridge. In addition, the amide bond between Tyr1 and Gly2 has been replaced by a methylene ether isostere and Gly3 has been deleted. The two linear analogues both contain the methylene ether isostere instead of the Tyr1-Gly2 amide bond and the shorter of the two lacks Gly3. The three compounds, and a beta-turn mimetic analogous to the 7-membered turn mimetic but with Gly3 included, were evaluated for specific binding to micro- and delta-opioid receptors in rat brain membranes. With the exception of the beta-turn mimetic the three other Leu-enkephalin analogues all bound with varying affinity to the micro- and delta-opioid receptors. From the results it could be concluded that Leu-enkephalin binds in a turn conformation to the opiate receptors, but that this conformation is not a (1-4) beta-turn.     

Dentin Phosphoprotein Mimetic Peptide Nanofibers Promote Biomineralization

Dentin phosphoprotein (DPP) is a major component of the dentin matrix playing crucial role in hydroxyapatite deposition during bone mineralization, making it a prime candidate for the design of novel materials for bone and tooth regeneration. The bioactivity of DPP‐derived proteins is controlled by the phosphorylation and dephosphorylation of the serine residues. Here an enzyme‐responsive peptide nanofiber system inducing biomineralization is demonstrated. It closely emulates the structural and functional properties of DPP and facilitates apatite‐like mineral deposition. The DPP‐mimetic peptide molecules self‐assemble through dephosphorylation by alkaline phosphatase (ALP), an enzyme participating in tooth and bone matrix mineralization. Nanofiber network formation is also induced through addition of calcium ions. The gelation process following nanofiber formation produces a mineralized extracellular matrix like material, where scaffold properties and phosphate groups promote mineralization. It is demonstrated that the DPP‐mimetic peptide nanofiber networks can be used for apatite‐like mineral deposition for bone regeneration.     

Discovery of dipeptidyl peptidase Ⅳ(DPP4) inhibitors based on a novel indole scaffold

Dipeptidyl peptidase Ⅳ(DPP4) inhibitors are proven in the treatment of type 2 diabetes.We designed and synthesized a series of novel indole compounds that selectively inhibit the activity of DPP4 over dipeptidyl peptidase 9(DPP9)(200 fold).We further co-crystallized DPP4 with indole sulfonamide(compound 1) to confirm a proposed binding mode.Good metabolic stability of the indole compounds represents another positive attribute for further development.     

A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening

The prevalence of type 2 diabetes is increasing dramatically throughout the world. Recently, dipeptidyl peptidase 4 (DPP4) was identified as a potential antidiabetes target. Many DPP4 inhibitors, such as sitagliptin and vildagliptin, have been developed and marketed, but superior therapeutic agents are still required. Therefore, we have developed new methodology for screening of DPP4 inhibitors. Absorption-based measurements with para-nitroaniline or fluorescence-based measurements with the coumarin derivative 7-amino-4-methylcoumarin are often used for the screening of protease inhibitors, including DPP4 inhibitors, but these strategies are not sufficiently sensitive because of interfering background absorption and fluorescence, thus giving rise to many false-positive and false-negative results. Therefore, we have designed and synthesised a novel DPP4 probe (Gly-Pro-BCD-Tb; Gly=glycine, Pro=proline, andBCD defines the backbone of the probe comprising an aniline derivative as on/off switch, a 7-amino-4-methyl-2(1H)-quinolinone (cs-124) as antenna moiety, and a diethylenetriamine-N,N,N',N',N'-pentaacetic acid (DTPA) as chelator moiety, Tb=terbium) for time-resolved fluorescence (TRF) measurements. TRF measurements with Gly-Pro-BCD-Tb showed high sensitivity and reliability in the inhibitory assay relative to Gly-Pro-MCA (MCA=4-methylcoumarin-7-amide), a conventional fluorescence probe for DPP4. Further, we employed our probe for high-throughput DPP4 inhibitor screening with 3841 randomly selected compounds and found that epibestatin, an epimer of bestatin (a well-known anticancer drug and general aminopeptidase inhibitor), showed dose-dependent DPP4 inhibitory activity. Interestingly, bestatin did not exhibit DPP4 inhibitory activity. We believe that this screening system will be useful for the discovery of DPP4 inhibitors with novel structural scaffolds.     

新型京尼平衍生物的合成、表征及抗糖尿病活性

为了寻找新型的抗糖尿病分子,从京尼平出发,经过甲基化（苄基化）、甲磺酰基取代、叠氮基取代和还原等4步反应得到含有活泼胺基的2个母核化合物. 这2个母核化合物与不同的酰氯反应,共得到13个新的京尼平衍生物,总收率41%~63%,其结构经核磁共振、质谱和元素分析确认. 活性测试结果表明,所有目标化合物在10 mmol/L 浓度下都有一定的抗糖尿病活性,其中化合物10f对二肽基肽酶4（DPP Ⅳ）的抑制率达31.2%.     

胰高血糖素样肽-1类似物的合成及其生物学活性研究

通过改变胰高血糖素样肽-1 (GLP-1)易被酶水解部位, 采用Fmoc/t-Bu正交保护固相合成策略, 并运用微波照射促进高效快速地合成了抗二肽基肽酶IV (DPP IV)的GLP-1类似物, 最后经过反相制备高效液相系统纯化得到目标多肽纯品. 生物学活性研究结果显示, 改造后的GLP-1类似物能有效抵抗DPP IV的水解, 并且有很好的降血糖活性. 所合成的GLP-1及其类似物的分子量和纯度均经过电喷雾质谱和高效液相确证.     

Design,Synthesis and SAR Studies of Novel and Potent Dipeptidyl Peptidase 4 Inhibitors

Dipeptidyl peptidase 4 (DPP‐4) is a clinically validated target for the treatment of type 2 diabetes mellitus (T2DM). To discover novel and potent DPP‐4 inhibitors, three series of compounds were designed and synthesized in this study based on our previously identified novel scaffold of 2‐phenyl‐3,4‐dihydro‐2H‐benzo[f]chromen‐3‐amine. Among the designed compounds, 41d‐1 was the most potent one with an IC₅₀ value of 16.00 nM. Besides, 41d‐1 (5 mg/kg) displayed a moderate glucose tolerance capability in ICR mice. Structure‐activity‐relationship (SAR) studies were discussed in detail, which is constructive for our further optimization.     

Discovery of novel inhibitors of anti-apoptotic Bcl-2 proteins derived from Bim BH3 domain

The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2 family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5 (K_D=9.48 nmol/L for Bcl-2) and SM-6 (K_D=0.08 nmol/L for Bcl-x_L), which exhibited improved binding affinity compared with the lead Bim (K_D=16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-x_L, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.