1. 研究目的与意义
Objectives: Peptides with the sequence of KKFVDLKKIANIL-NH2 from cinobufagin venom toad need to be synthesized with the utilization of solid phase chemical synthesis, and with the index of the minimum inhibitory/bactericidal concentrations (MICs/MBCs), antibacterial efficacy of the peptides is going to be examined.Significance: Nowadays, many serious diseases, like tuberculosis or tetanus, are caused by pathogenic bacteria. Moreover, because of unfavorable economic factors and regulatory challenges in gaining approvals, the development of novel antimicrobial agents has stagnated for many years. Therefore, the research on antimicrobial agents allows of no delay. At present, antibiotics are the most widely used agents to combat bacteria while many bacteria have generated resistance to it. According to the previous experimental findings, antimicrobial peptides (AMPs) are widely considered as the most potential alternatives to antibiotics. Therefore, we choose AMPs to study. With the intention to improve the antibacterial efficacy, reduce the toxicity, increase the stability and resistance to drug resistance, we modified the peptides to the sequence of-KKFVDLKKIANIL-NH2 for the following assays. Through the whole experiment, it is a great improvement and a challenge for me to study the methods and operation standards of antimicrobial peptides synthesis and identification which I have never learned before.
2. 文献综述
The Synthesis and Activity of Antimicrobial Peptides from Cinobufagin Venom ToadLin YaxianNanjing University of Chinese MedicineAbstractNowadays, antibiotics are the most widely used medicine to treat diseases caused by pathogenic bacteria. However, because the speed of antibiotics development is much slower than that of drug resistance generated by bacteria, we urgently need to exploit effective antimicrobial agents. According to previous experimental results, antimicrobial peptides (AMPs) from cinobufagin venom toad have been universally considered as a promising alternative for antibiotics. Therefore, after searching and analyzing relevant documents, this article summarizes the methods to synthesize AMPs, antimicrobial mechanism and activity assays in vitro which have been verified. Key words: Antimicrobial peptides (AMPs); Synthesis; Antimicrobial ActivityIntroductionThe Antimicrobial peptides (AMPs) we plan to research on is from a traditional Chinese medicine called cinobufagin venom toad, which is a secreta from the poison gland of frog skin. Cinobufagin venom toad. Cinobufagin venom toad has the traditional function of detoxifying and relieving pains, is utilized in furunculosis, ulcer or other intractable diseases. AMPs are an important part of innate immune system, which play important roles on protecting hosts from invading pathogenic bacteria and a wide range of organisms and have been considered as first line of defense (Nguyen, Haney and Vogel, 2011; Jenssen, Hamill and Hancock, 2006). Since the first AMP was found from insects and animals in 1980s, more than 2500 AMPs have been deposited in the Antimicrobial Peptides Database (Zhang et al., 2016). Although AMPs have a broad-spectrum antimicrobial activity and high efficiency, there are some inherent defects hindering the development of them, for example, high toxicity against mammalian cells, poor stability under physiological salt concentration and serum conditions and so on (Wang et al., 2015). With the intention to modify clinically available AMPs, this article summarized the methods to the synthesis of AMPs, and the assays to examine their antimicrobial efficiency.Methods1. Antimicrobial Peptides Synthesis1.1 Modes of Production To do the research on AMPs, the first thing we need to do is to obtain the peptides. The peptides can be produced in the following ways:1.1.1 Enzymatic hydrolysisThe most common way to product peptides is enzymatic hydrolysis to natural protein sources with proteolytic enzymes, like pepsin or trypsin (Korhonen and Pihlanto, 2006). The advantage of this method is that the product yields are very high and enzymes are low-cost and safe (Hernndez-Ledesma, del Mar Contreras and Recio, 2011).1.1.2 Fermentation and maturation processPeptides can be generated by starter and non-starter bacteria used in the manufacture of fermented dairy products which have shown beneficial effects on blood pressure in several rat models and human studies (Hernndez-Ledesma, del Mar Contreras and Recio, 2011).1.1.3 Recombinant DNA techniques Recombinant DNA techniques have been experimented to product specific peptides. The challenges of it is the susceptibility of peptides to degradation by proteases or peptidases and that the products may be harmful to the host (Hernndez-Ledesma, del Mar Contreras and Recio, 2011).1.2 Methods of Peptides SynthesisThe methods to synthesize peptides mainly include solid-phase peptides synthesis (SPPS) and liquid-phase peptides synthesis (LPPS).1.2.1 Solid-phase peptides synthesis (SPPS)Solid-phase peptides synthesis is the most widely used method for synthesizing peptides. After modifying peptides sequence and weighing the amino acids required, we can combine amino acids with resin through coupling reaction. Then, the resin and Fmoc on the side chain can be cut through cleavage. Consequently, the peptides modified can be received. However, deeper investigation has shown that the individual coupling steps are not quantitative, so that erroneous sequences accumulate, together with the desired polypeptides. Another disadvantage of SPPS is the restriction in the choice of solvents, coupling methods and protecting groups (BAYER and MUTTER, 1974).1.2.2 Liquid-phase peptides synthesis (LPPS)To overcome the disadvantages of SPPS, people have developed another method to synthesize peptides: liquid-phase peptides synthesis. Liquid-phase synthesis which basically consists of the following: a soluble, linear homopolymer serves as the C-terminal protecting group for the peptide which is to be synthesized. Mono or bifunctional polymers such as polyethylene glycol have proved themselves as being especially good. All reactions are carried out under homogeneous conditions but contrary to the classical method, the activated components can be used in large excess so that quantitative coupling is achieved. The yields from the individual coupling steps can then be determined using simple tests (Bayer et al., 1972).2. Antimicrobial Activity Assay2.1 Antimicrobial MechanismAt present, the antimicrobial mechanism of AMPs is still not very definite. Many AMPs work on bacterial membranes or generalized targets which is different from antibiotics. Previous studies have demonstrated several possible explanations for the antimicrobial mechanism of AMPs: Because of distinct membrane-bound amphipathic conformation and cationicity, AMPs can disrupt the integrity of bacterial membrane in many ways, such as carpet model or toroidal pore. They can also target key cellular process including DNA and protein synthesis, protein folding and cell wall synthesis. Some AMPs can give play to direct antimicrobial activities themselves (Nguyen, Haney and Vogel, 2011).2.2 In Vitro Activity AssayThe first thing to test novel AMPs is to examine their efficacies in vitro so that we can modify AMPs according to the disadvantages through the test, like poor stability in serum condition or high toxicity. The approaches to modify AMPs includes the methylation to some amino acids in peptides, cycling AMPs or so on. The bacteria used in this experiment should be include gram-negative bacteria (like E. coli), gram-positive (like S. aureus) and fungus (like C. albicans). Antimicrobial efficacy is usually expressed with the minimum inhibitory/bactericidal concentrations (MICs/MBCs), and the geometric mean (GM) values of them (Wang et al., 2015).The minimum inhibitory/bactericidal concentration of each peptide was determined using a broth micro-dilution assay modified from the method of Hancock. Further dilutions of the stock solution to 10 times the required test concentration were made to reach a final concentration of 5105 colony-forming units (CFU)/mL. the MIC/MBC of each peptide was read as the lowest concentration of peptide that inhibited visible growth of the bacteria after 24h incubation at 37℃.All MICs/MBCs determinations were performed in duplicate, and are the average of three independent determinations. (Ryge et al., 2004)2.3 In Vivo Activity AssayMost AMPs have great activity in vitro but no activity in vivo. Therefore, antimicrobial efficacy test in vivo is a significant step in the discovery of novel AMPs. We can inject AMPs in mouse or other infectious animal models to combat bacteria with the index of mortality rate and colony-forming units (CFU) of bacteria. Besides, the most basic requirement for drugs is safety so that we should verify the toxicity of AMPs through the activity assay in vivo.Peptides can be evaluated for their efficiency in a mouse peritonitis model, where the intraperitoneal (i.p.) dose of bacterium which led to establish a widespread bacterial infection by 24 h. At 0.5, 2,4, and 8 h post-infection, the mice were treated with physiological saline solution (control), 5 mg/kg peptides or 5 mg/kg gentamicin. We also tested the toxicity of the different concentrations of peptides in vivo. 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, respectively (Wang et al., 2015).AMPs have only been tested in clinical trials relatively recently, and to date, none have received US Food and Drug Administration (FDA) approval, with the exception of gramicidin for topical administrations. Magainin Pharmaceuticals provided early high hopes for the field, with impressive data in early Phase I and II clinical trials. Currently, there are only a small number of companies researching AMPs as therapeutics, but there are at least 10 AMP-derived compounds in varying stages of clinical development (Taylor 2304[5] Korhonen, H. and Pihlanto, A. (2006). Bioactive peptides: Production and functionality. Elsevier 16,945-960[6] Hernndez-Ledesma, B., del Mar Contreras, M. and Recio, I. (2011). Antihypertensive peptides: Production, bioavailability and incorporation into foods. Elsevier 165,23-35[7] BAYER, E. and MUTTER, M. (1972). Liquid Phase Synthesis of Peptides. Nature 237,512-513[8] Bayer, E., Mutter, M., Uhmann, R., Polster, J. and Mauser, H. (1974). Kinetic studies of the liquid phase peptide synthesis. Journal of the American Chemical Society 96,7333-7336[9] Ryge, T., Doisy, X., Ifrah, D., Olsen, J. and Hansen, P. (2004). New indolicidin analogues with potent antibacterial activity*. Peptide Res. 64, 171185[10] Taylor Francis. (2013). Antimicrobial peptides: new drugs for bad bugs
3. 设计方案和技术路线
First, solid-phase synthesis method was used to repeat the addition of precisely-named amino acids required for synthesis from the oxime end (carboxyl group) to the oxime end (amino group). Because the NH2 is the C-terminus of polypeptides, we choose Rink Amide MBHA Resin to synthesize the peptide. In order to avoid the side-reaction of resin and amino acid (aa) residues, both the amino of resin and the side-chain of amino acid residues are protected by the F-moc. After the deblocking of the F-moc and the activation of aa, we synthesized the polypeptide from N-terminal end to C-terminal end one by one with the use of Tribute peptide synthesizer.Then, we made the use of HPLC to purify the peptides and LC-MS to verify the purity and obtain the molecular weight (MW) and the sequence- KKFVDLKKIANIL-NH2.At last, we conducted the antimicrobial activity screening against three types of bacteria, including S. aureus (Gram-positive bacteria), E. coli (Gram-negative bacteria) and C. albicans (Fungus) to gain the inhibitory/bactericidal concentrations (MICs/MBCs). Therefore, we can evaluate the antibacterial efficacy of the engineered peptide.Technical route: Consolidate basic knowledge → Review literature → Synthesize peptides → Sequence identification → Functional screening(Antimicrobial experiments)
4. 工作计划
TIME CONTENT PROGRESS RATE2022.03.03~03.08 Read articles regarding to the experiment and do the presentation completed2022.03.09~03.23 Do the experiment completed2022.03.24~04.04 Read relative articles and write the initiating report and the review paper 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
Nowadays, we put most of our effect on the study of botanical drug in the Chinese medicine field. Since animal medicine plays an important role in disease treatments, the research on it is far from enough. Peptides have been found from the poison gland of the frog skin which was named cinobufagin venom toad in Chinese Medicine and peptides have a small molecular weight and effect on bacterial cell membranes. Therefore, we choose peptides to do our research.At present, the most effective medicine in the clinic is antibiotics while many bacteria are resistant to it. For this reason, we chose three types of bacteria, including gram-positive, gram-negative bacteria and fungus, to examine peptides antimicrobial efficiency against them, with the intention of finding a potential alternative for antibiotics.
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