1. 研究目的与意义
Antimicrobial peptides (AMPs), have been discovered from many insects and animals initially, which are ubiquitous in nature. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as promising alternatives for antibiotics and many peptides from eukaryotic organism act on bacterial membranes or other generalized targets, which makes it an advantage of AMPs because developments of microbial resistance by gene mutation is less likely. AMPs have played a fundamental role in natural immunity, which possess the features of broad spectrum, high efficiency, selective toxicity, stability and high proteolytic resistance. At present, different types of antimicrobial peptides have been isolated and identified from various organisms including bacteria, fungi, plants, insects, amphibians, fish, birds, mammals, and humans.
2. 文献综述
Introduction of Peptide synthesis and antimicrobial peptideLiu SiyanNanjing university of Chinese medicineBackgroundIncreasingly appearance of drug-resistant or multidrug-resistant pathogens causes multiple antibiotic-resistant infections, conventional antibiotics are becoming useless. AMPs are considered as promising alternatives to antibiotics. AMPs were discovered from natural sources at first, and then people started synthesizing peptide analogs, after that, de novo peptide design appeared, also, they are being discovered through sequences in large proteins.Peptide synthesisPeptide is the production of peptides synthesis where multiple amino acids are linked via amide bonds. Chemical synthesis of peptides can be carried out using classical solution-phase techniques, however, SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support, so it has become a universal method now. Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate protecting groups such as Boc (acid-labile) or Fmoc (base-labile) and the N-terminal of the amide resin also should have protecting group (PG) to prevent undesirable side reactions with the various amino acid side chains. The general SPPS procedure is one of the repeated cycles of alternate N-terminal deprotection and coupling reactions.At the end of the synthesis, the crude peptide is cleaved from the solid support while simultaneously removing all protecting groups using a reagent strong acid like trifluoroacetic acid (TFA). The crude peptide can be precipitated from a non-polar solvent like diethyl ether in order to remove organic soluble by products. In the end, the polypeptide was synthesized.Antimicrobial peptides（AMPs）Strengths and weaknesses of antimicrobial peptides (AMPs)AMPs have been discovered from insects and animals initially and are common in nature, which means they are important in organisms intrinsic immune system and AMPs obtained from higher eukaryote organisms have additional immunomodulatory activities (Hancock and Sahl, 2006). Also, AMPs can act as hormones, growth factors, ion channel ligands, anti-infectives and so on (Fosgerau and Hoffmann, 2015). AMPs usually have rapid and broad spectrums activity against pathogens including bacteria, fungi and parasite. Furthermore, compared with conventional antibiotics, which usually target specific proteins, their nonspecific membrane penetration reduces the microbial resistance by gene mutation. However, some inherent defects do exist such as poor stability under, high toxicity, low resistance to protease degradation (Wang et al., 2019), short circulating plasm half-life and so on. Moreover, the hydrophobicity is highly correlated with cytotoxicity and antimicrobial activity, which means a good balance should be kept. Mode of action of antimicrobial peptides（AMPs） Generally, antimicrobial activities of AMPs are relevant to several structure features such as conformation, charge, hydrophobicity, amphipathicity and polar angle (NY, 2003). Among them, the most relative features are cationic charge and hydrophobicity, the former property promotes selectivity for negatively charged microbial cytoplasmic membranes over zwitterionic mammalian membranes, whereas the latter facilitates interactions with the fatty acyl chains (Nguyen, Haney and Vogel, 2011). Besides, there are two largest groups of the conformation: α-helical and β-sheet. The α-helical antimicrobial peptides are abundant in the extracellular fluids of insects and frogs and frequently exist as extended or unstructured conformers in solution. Many of these peptides only become helical upon interaction with amphipathic phospholipid mem- branes. The β-sheet peptides represent a highly diverse group of molecules at the level of primary structure. Despite such differences, these peptides share common features, including amphipathic composition, with distinct hydrophilic and hydrophobic surfacES (NY, 2003). Although the most common mode of action is disrupting the integrity of membrane, there are some other modes involve targeting key cellular processes (Nicolas, 2009). Besides, there are many correlations between the conformation, polar angle and hydrophobicity, normally, higher helicity and smaller polar angle (θ) contribute to higher sterilization rate and higher cytotoxicity (Uematsu and Matsuzaki, 2000). However, inducing proper folding and kink in the middle of the α-helix can reduce the cytotoxicity (Nguyen, Haney and Vogel, 2011).Different assays to test peptidesThe peptides were normally first assessed for their antibacterial efficiencies in killing various infectious Gram-negative bacteria (like E.coli), Gram-positive pathogens(like S.aureus) and some kind of fungi(like M.albican) ,The antibacterial efficacies of the engineered peptides against bacterial strains were expressed with the minimum inhibitory/ bactericidal concentrations (MICs/MBCs), and the geometric mean (GM) values of the MICs/MBCs of the peptides across all of the tested bacterial strains represents the average bacteriostatic/bactericidal level of a peptide. And then, the biocompatibility, salt, serum, and protease sensitivities and cytotoxicities were also evaluated in vitro. AMPs exert antimicrobial activity via a non-receptor-mediated membrane permeation mechanism, which possibly reduced the biocompatibility of AMPs to normal cells. High biocompatibility is a prerequisite for AMPs to break through clinical application bottlenecks and some peptides have hemolytic activities that cause to low biocompatibility so it must be tested. As for salt and serum. The susceptibility of AMPs to high salt concentrations has always been an inevitable problem in their clinical application; for example, human β- defensin 1 has been found to lose its activity against P. aeruginosa in airway surface fluids with high salt concentration from cystic fibrosis bronchial xenografts, and hence, high salt resistance is an essential feature for the engineered peptides. Then, poor proteolytic resistance has always been an insurmountable obstacle in the clinical application of AMPs as higher proteolytic resistance contributes to a better efficacy of peptides.so this is one the most important factor to be tested. Finally, in vivo efficacies of the peptides were measured. Most AMPs have potent antibacterial activity in vitro but no activity in vivo. Also, the toxicity of the different peptides and different concentrations of the peptide should be tested in vivo,for example，blood urea nitrogen (BUN) and aminotransferases have been long regarded as the sensitive indicators of renal and hepatic toxicity of drugs in humans and animals (Rykaczewska et al., 2018，Kim et al., 2018) respectively. referencesWang, J., Song, J., Yang, Z., He, S., Yang, Y., Feng, X., Dou, X. and Shan, A. (2019). Antimicrobial Peptides with High Proteolytic Resistance for Combating Gram-Negative Bacteria.Nguyen, L., Haney, E. and Vogel, H. (2011). The expanding scope of antimicrobial peptide structures and their modes of action.NY, Y. (2003). Mechanisms of antimicrobial peptide action and resistance. Fosgerau, K. and Hoffmann, T. (2015). Peptide therapeutics: current status and future directions.Nicolas, P. (2009). Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides.Uematsu, N. and Matsuzaki, K. (2000). Polar Angle as a Determinant of Amphipathic α-Helix-Lipid Interactions: A Model Peptide Study.Hancock, R. and Sahl, H. (2006). Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.Rykaczewska, A., Gajcka, M., Dbrowski, M., Winiewska, A., Szczeniewska, J., Gajcki, M. and Zielonka, . (2018). Growth performance, selected blood biochemical parameters and body weights of pre-pubertal gilts fed diets supplemented with different doses of zearalenone (ZEN).Kim, W., Zhu, W., Hendricks, G., Van Tyne, D., Steele, A., Keohane, C., Fricke, N., Conery, A., Shen, S., Pan, W., Lee, K., Rajamuthiah, R., Fuchs, B., Vlahovska, P., Wuest, W., Gilmore, M., Gao, H., Ausubel, F. and Mylonakis, E. (2018). A new class of synthetic retinoid antibiotics effective against bacterial persisters.
3. 设计方案和技术路线
Firstly, peptides should be synthesized through the solid-phase peptide synthesis method, which allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support. In the SPPS process, we use an amide resin and the N-terminal protecting group (PG) which is Fmoc. Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate protecting groups such as Fmoc. The first step of SPPS is to do the deblocking of resin and activation of mythe last amino acid (L) in the sequence, then the next procedure is repeated cycles of N-terminal deprotection and coupling reactions. This cycle repeats until the desired sequence has been synthesized.At the end of the synthesis, the crude peptide is cleaved from the solid support while simultaneously removing all protecting groups using a reagent strong acid like trifluoroacetic acid (TFA). The crude peptide can be precipitated from a non-polar solvent like diethyl ether in order to remove organic soluble by products. In the end, the peptide was synthesized. Secondly, the identification and analysis of the peptide should be done using High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), so that the molecular weight and the primary structure can be measured. As for the secondary structure, we may use the bioinformatics approach to evaluate. Finally, through the relevant antibacterial experiments, the function of the peptide will be identified. In antibacterial experiments, we tested the minimum inhibitory concentrations (MIC) to express the antibacterial efficacies of peptides on E. coli, S. aureus, and C. albicans. Technical route: Review literature--- Peptide synthesis--- Identification and analysis---Antimicrobial experiments (functional screening)
4. 工作计划
2022.03.03~03.08 Read literature regarding to the experiment, attending class to learn some knowledge about AMPs completed2022.03.09~03.23 Do the experiment To be completed2022.03.24~04.04 Read relative articles and write the initiating report and the review paper To be completed2022.04.05~04.15 Finish the experiment To be completed2022.04.16~05.15 Analyze the experimental data and write the research paper To be completed
5. 难点与创新点
According to some literatures, the mechanism of microbicide mechanism is membrane destruction and diverse lengths and subtypes of hydrophobic/cationic amino acids had a considerable influence on the cell selectivity and the proteolytic resistance. And in my sequence, amino acids were rationally arranged to enhance its bactericidal activity and proteolytic resistance. Lys, which havehas sufficient cationicity, was selected to construct a hydrophilic core, while placing Pro at the C-terminus of Lys is towill completely block Lys from cleavage by protease like trypsin. Additionally, proline-induced folding or kink in the middle of the α-helix can help peptides remain antimicrobial activity but have low cytotoxicity. Furthermore, Ile, Leu and Val with long aliphatic side chains were preferentially placed at both termini of the sequence unit to increase the hydrophobic face depth and to avoid degradation by protease like chymotrypsin. However, since hydrophobicity is highly correlated with cytotoxicity, so we need to achieve an appropriate balance between hydrophobicity, cytotoxicity and activity, which means we should further explore the optimal cell selectivity.
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