摘要
抗生素因抗菌效果显著得以在临床上广泛应用乃至滥用,致使大量细菌对其产生耐药性。抗生素本身是细菌为保护其免受病原菌攻击而产生的一类化学物质,因而病原菌也能通过产生水解酶或药泵、基因突变、改变细胞膜性质等方式形成耐药。目前,耐药菌已遍布全球,严重威胁人类健康。尽管科学家针对耐药菌不断改进抗生素,但细菌总能以更聪明的方式形成耐药,使得抗生素的开发难度与日俱增。抗菌肽(AMP)是生物体为抵御外源性病原菌入侵而产生的一类多肽物质,也是自然免疫的重要组成部分。由于抗菌肽的作用机制与传统抗生素存在显著区别,因而具有抗菌谱广、杀菌快速、耐药性低等传统抗生素无法比拟的优点,有望开发成为一类高效、低毒的新型抗菌药物。
鉴于抗菌肽的固有特点及其广阔的应用前景,研究人员基于抗菌肽的阳离子性和两亲性结构特征,通过序列模板法和组合肽库法设计并筛选获得了多个具有较好活性的抗菌肽。然而,规模化生产成本高、耐酶稳定性差、潜在溶血毒性是制约抗菌肽进入实际应用的瓶颈。小分子抗菌拟肽及其类似物的研究使其向实际应用迈进了一大步,然而天然抗菌肽与其类似物仍存在抗菌活性不理想及潜在溶血毒性等问题。目前,由于没有可靠的理论模型用于抗菌活性与溶血毒性的准确预测,因而难以从理论上指导抗菌肽的设计与筛选,极大增加了抗菌肽的理论设计与应用开发难度。因而,通过建立理论预测模型指导抗菌肽的合理设计与虚拟筛选,有望发现抗菌活性好、耐酶解能力强、溶血活性低的小分子抗菌肽,可为低耐药性抗菌药物的应用开发奠定理论与物质基础。综上所述,本文基于定量构效关系(QSAR)研究建立抗菌肽的理论预测模型,指导基于天然氨基酸的抗菌五肽及抗菌四肽的组合设计与虚拟筛选,最后通过抗菌活性与溶血毒性实验验证理论模型的可靠性,取得了如下研究结果：
1.本文使用来自数据库的89种氨基酸物理化学性质用于抗菌肽的结构表征,并使用逐步回归(STR)与多元线性回归(MLR)相结合的方法(STR-MLR)成功建立了系列合成抗菌肽(R2=0.793,Q2=0.751)、novispirin抗菌肽(R2=0.970,Q2=0.872)和表面粘附抗菌肽(R2=0.686,Q2=0.639)的QSAR模型。本文所建QSAR模型的稳定性与预测能力与文献相当甚至更优。STR-MLR模型的标准化系数不仅可用于分析抗菌肽的优势位点,而且能用于计算氨基酸贡献值以发现优势氨基酸,进而指导抗菌肽的理论设计或结构改造。STR-MLR模型具有稳定性好、预测能力强、物理化学意义明确、操作简便、易于实现等优点,可有效用于抗菌肽的虚拟设计与高效筛选。
2.本文使用STR-MLR方法分别建立了六肽和五肽对大肠杆菌(E. coli)和金黄色葡萄球菌(S. aureus)抗菌活性的QASR模型,通过模型标准化系数并结合保守残基分析,发现了抗菌六肽和五肽的优势位点与优势氨基酸。同时,使用比较分子力场分析(CoMFA)与比较相似性指数分析(CoMSIA)方法分别建立了该系列抗菌肽基于N-端和C-端公共骨架叠合的三维构效关系(3D-QSAR)模型。CoMFA与CoMSIA对E. coli和S. aureus抗菌活性所建模型的交互检验复相关系数均高于0.400,表明所建模型具有较好的稳定性与预测能力；而且,CoMFA与CoMSIA等值面图可为抗菌肽的合理设计及结构改造提供可视化信息。本文基于小分子抗菌肽的STR-MLR模型及3D-QSAR研究结果,以牛乳铁素片段LfcinB6(RRWQWR)为模板,结合抗菌肽的阳离子性、两亲性、优势位点、优势氨基酸等信息,确定抗菌肽的虚拟组合设计方案,并据此虚拟设计540条理论抗菌五肽。首先根据阳离子性与两亲性的结构特征初步筛选121条抗菌肽,然后使用STR-MLR、 CoMFA、CoMSIA模型预测并筛选出4条理论抗菌肽,最后通过抗菌活性与溶血毒性实验验证抗菌肽理论设计与筛选方法的可靠性。此外,为了探索序列更短的多肽是否仍具有抗菌活性,本文基于LfcinB6、结合保守残基分析与QSAR研究,定向设计一条抗菌四肽进行实验研究。
3.本研究分别使用使用固相合成方法合成、反相高效液相色谱(RP-HPLC)纯化及质谱(MS)鉴定已筛选的抗菌五肽,高效液相色谱(HPLC)分析结果显示,固相合成抗菌肽的纯度达到95%以上,质谱测定与理论计算分子质量完全相符,达到抗菌活性与溶血毒性实验研究的要求。
4.本文为验证已筛选抗菌肽的抗菌效果,首先通过琼脂平板法测定抗菌肽对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单胞菌的生长抑制作用,然后使用微量稀释法定量测定抗菌肽的最小抑菌浓度(MIC)。琼脂平板实验结果表明除"RWQWR"外,其他抗菌肽均具有较模板肽(LfcinB6)优异的抗菌活性,而且抑菌斑在7天内不缩小,表明抗菌肽的抗菌效果较为持久。抗菌肽对比实验显示,设计抗菌肽的最佳抗菌活性仅为庆大霉素的50%左右,因而还有待进一步提高抗菌活性。抗菌活性定量测定结果表明,设计抗菌肽对4种细菌的MIC在16-128μg·ml-1之间,与文献报道小分子抗菌肽的抗菌活性基本相当,有必要在此基础上通过结构修饰等方法进一步提高其抗菌活性,进而提高其实际应用潜力。对比实验测定与模型预测抗菌活性发现,三种定量预测模型均可较准确的预测抗菌肽活性。总体而言,CoMFA模型对抗菌活性的预测能力最优,而STR-MLR模型的预测能力稍差,可能与STR-MLR建模样本数较少有关。
5.溶血毒性是抗菌肽能否进入实际应用的重要前提,本研究分别使用平板法与微量稀释法测定设计抗菌肽的溶血活性。平板实验显示设计抗菌肽在72小时不出现溶血现象,相比48小时就出现溶血的模板肽(LfcinB6),设计抗菌肽的溶血活性并不明显。微量稀释法所测定的抗菌肽溶血百分率显示,设计抗菌肽在100μg·ml-1时才出现溶血效应,且浓度在1mg·ml-1时的溶血率均低于5%,表明设计抗菌肽具有良好的安全性,具有进一步开发利用的潜力。
6.本文设计抗菌四肽对4种菌株的MIC在8-64μg·ml-1之间,表明更小分子的抗菌肽同样具有优异的抗菌活性,为设计与筛选化学合成容易小分子抗菌肽奠定了基础。然而,该抗菌肽在32μg·ml-1时出现溶血效应,且在1mg·ml-1时的溶血率百分率高达25%,因而有必要将其作为先导化合物进行结构改造,以进一步提高其安全性。
本文将生物信息学、化学信息学与生物化学有机结合,通过抗菌肽的QSAR研究以及保守残基分析,建立了抗菌肽设计与筛选的理论方案。根据抗菌六肽、五肽的二维及三维QSAR研究结果,结合抗菌肽的阳离子性与两亲性的结构特征,虚拟组合设计并高效筛选获得4个理论抗菌肽,最后通过抗菌活性及溶血毒性实验研究,获得3个抗菌活性较好、溶血毒性小的抗菌五肽。本文设计抗菌肽的理论预测与实验测定抗菌活性相当接近,说明基于QSAR理论预测模型的抗菌肽设计与筛选方法高效、可靠、易行。本研究不仅成功建立了抗菌肽理论设计、高效筛选与实验验证的完整技术方案,而且筛选获得3个小分子抗菌肽先导物,为抗菌肽的理论研究与应用开发奠定了良好基础。
Antibiotics are widely used and abuse in clinic because its significant antimicrobial effect, which result in the resistance of lots of bacteria. Antibiotics are the chemical substance generated by bacteria in order to avoid attacking from pathogenic microorganism. However, the microorganism can become resistance through hydrolase, drug pump, genetic mutation or membrane transformation. Currently, the bacterium with resistance is existed in anywhere and threatens the human health seriously. For bacteria with resistance, the antibiotics are improved by researchers, but they can become resistance more cleverly, and the development of antibiotics is more difficult. Antimicrobial peptide (AMP) is the polypeptide, which protected the organism avoiding attack from pathogenic bacteria, and it is the important component of innate system. The antimicrobial mechanism of AMP is significant distinction from antibiotics, thus it has broad antimicrobial spectrum, rapidly disinfection and low resistance, which has the potential to be developed as the novel antimicrobial agent with high efficiency and low toxicity.
The AMP becomes the focus because of its inherent characters and magnificent prospects. There are some AMPs are designed and screened through sequence motif and combinational peptide library method, which based on cationic and amphipathic properties of AMP. However, the AMP has some deficiency, such as high cost of manufacture, poor protease stability and potential hemolysis, which limited them into clinic application. The emerging of short AMP and its analogues promotes the clinic application of AMP greatly, but they have the problem of non-ideal activity and potential hemolysis. Currently, there is no reliable theory model for the prediction of antimicrobial and hemolytic activity, which makes the AMP design and development more difficult. Therefore, the theoretic model uses to guide the rational design and virtual screening of AMP, and the short AMP with high activity, good hydrolytic stability and low hemolysis can be explored, which may become the basis for development of novel antimicrobial agent. Consequently, this study constructs the theoretical model based on quantitative structure activity relationship (QSAR) to guide the combinational design and virtual screening of AMP, and the antimicrobial and hemolytic experiments are used to validate the reliability of theoretical model. The research results are gained in this study as following:
The AMP are characterized by89physicochemical properties of amino acids, and the method combined the stepwise regression and multiple linear regression (STR-MLR) is used to construct QSAR model for synthesis AMP (R2=0.793, Q2=0.751), novispirin AMP (R2=0.970, Q2=0.872) and surface-tethered AMP (R2=0.686, Q=0.639). The reliability and predictive ability of STR-MLR model is superior or equivalent to that constructed in literatures. The normalized regression coefficients (NRC) of STR-MLR can discover the dominant position, and the contribution of amino acid calculated by NRC can reveal important amino acids, which can guide the design and modification of AMP directly. STR-MLR model has many advantages, such as good reliability and predictive ability, definite physicochemical meaning, simply operation and facilitate realization.
The quantitative predictive models are established by STR-MLR and conserved residues analysis is performed for serial hexapeptides and pentapeptides with antimicrobial activity for Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), which discovers the advantage positions and dominant amino acids. Based on the alignment of common backbone of N-and C-terminal, the three dimension quantitative structure activity relationship (3D-QSAR) models are constructed by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) for short AMP. The correlation coefficient (Q2) of cross validation is larger than0.400, meaning the3D-QSAR model has good reliability and predictive ability, which can guide the design and screening of AMP. Additionally, the counter map of CoMFA and CoMSIA can provide the visible information for molecular design and modification, which can guide the combinational design de residue modification of AMP. The scheme of virtual combination design is defined based on the STR-MLR model and3D-QSAR results, template peptide (LfcinB6, RRWQWR), cationic and amphipathic properties, advantage positions and dominant amino acids, then540pentapeptides are generated and121peptides are screened according to cationic and amphipathic properties of AMP. Finally, the design and screening method of AMP are validated by antimicrobial and hemolytic experiments of4pentapeptides, which predicted and screened by STR-MLR, CoMFA, CoMSIA models comprehensively. Additionally, in order to explorer the antimicrobial activity of shorter peptide, a tetrapeptide is designed for experimental research base sequence analysis and QSAR results.
The designed AMP is synthesized, purified and analyzed by solid synthesis, reverse phase high performance liquid chromatography (RP-HPLC) and mass spectrum (MS) respectively. The purity of AMPs is higher than95%, and the molecular mass is equivalent to that determined by MS, which meet the requests of antimicrobial and hemolytic experiments.
In order to validate the antimicrobial efficiency, the growth inhibition and minimal inhibition concentration (MIC) for E. coli, S. aureus, Bacillus subtilis and Pseudomonas aeruginosa are determined by agar plate assay and broth microdilution method respectively. The agar plate assay indicates that AMPs have superior activity than template peptide except "RWQWR", and the blot of AMP is not shrinked in7days, which means the AMPs have stable activity. Comparing to the antibiotics, the inhibition efficiency of AMP is only half of gentamycin, which should be improved greatly. The MIC of designed AMP for4strains is between16and128μg·ml-1, which should be improved through structural modification. Comparison between determined and predicted activity, three QSAR models built here can predict the antimicrobial activity accurately. In general, the CoMFA model has best predictive ability, but the STR-MLR mode is not ideal, which attributes to the few samples used to establish QSAR model.
The hemolysis of AMP is the prerequisite of its application, and it is evaluated by plate assay and microdilution method respectively. The plate assay shows the AMPs have not hemolytic effect in72hour, which is superior to that of LfcinB6. The percentage hemolysis of AMP determined by microdillution method indicates they have hemolytic effect with concentration is100μg·ml-1,but the percentage hemolysis below5%with concentration is1mg·ml-1. The experimental result shows the AMP has good safety and potentiality for development.
The MIC of tetrapeptide is8-64μg·ml-1for4strains, which indicate the short AMP with high activity and become the basis for design and screening of short AMP with easy synthesis. However, the tetrapetide has hemolytic efficiency with concentration is32μg·ml-1and the hemolytic percentage is up to25%when the concentration is1mg·ml-1, which shows it can be studied as lead compound and promoted the security through structural modification.
In ths study, the method combining bioinformatics, chemoinformatics and biochemistry are used to AMP research. The design and screening scheme of AMP is established based on QASR research and conserved residues analysis. Based2D-QSAR and3D-QSAR results, cationic and amphipathic properties of AMP, there are4pentapeptides are designed and screened for experimental validation. Finally,3pentapeptides with high activity and low hemolysis are validated by antimicrobial and hemolytic experiments. The determined and predicted activity is very close, which shows the method of design and screening based QSAR predictive model is effective and realibity for AMP development. Additionally, the scheme is simple, facilitate, high-performance and reliability. In general, the theoretical scheme of rational design, high-performance screening and experimental validation for AMP are established and3short AMP are screened, which become the basis for theoretial research and development of AMP.
引文
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