1. 研究目的与意义
Objective: Fmoc solid-phase peptide synthesis of a new sequence of peptide (KWPLIGGILKKVL-NH2) and the research of its antimicrobial activity are to be carried out.Significance: Antimicrobial peptides (AMPs) are small molecule peptides with broad spectrum antimicrobial activities, high efficiency and selective toxicity which exhibit potent function in natural immunity against bacteria, viruses and fungi. In addition to their fundamental roles, AMPs are also considered to be important in various functions such as angiogenesis and wound healing. With the increasing drug resistance to conventional antimicrobial agents, the promising AMPs with ability to kill multidrug-resistant microorganisms are shown to be an essential alternative to antibiotics. It is expected that more research of AMPs will be done to develop efficient treatments.
2. 文献综述
The strengths and growth potential of antimicrobial peptidesYin, DanjingSchool of Pharmacy, Nanjing University of Chinese MedicineAbstractIn the past few years, antimicrobial peptides (AMPs) have exhibited potent antimicrobial activities in natural immunity and gained heated discussion. When countless research projects are carried out to identify the principles and mechanisms governing characteristics of bioactive proteins and peptides, the promising AMPs with ability to kill multidrug-resistant microorganisms are shown to be an essential alternative to antibiotics. There have been considerable advances in the synthesis process of AMPs and diverse properties which are required for antimicrobial drug discovery.Keywords: Peptide design; Antimicrobial peptide; peptide synthesis; drug-resistant bacteria1 IntroductionAntimicrobial peptides(AMPs) are small molecule peptides with broad spectrum antimicrobial activities which exhibit potent function in natural immunity. In addition to their fundamental role, AMPs are also considered to be important in various functions such as angiogenesis and wound healing. It is expected that more research of AMPs will be done to develop efficient treatments. To deal with increasing drug- resistant pathogens, high selective and potent peptides are required to be designed. More attention needs to be focused on optimizing designed sequences and formulation development. Rational design of synthetic AMPs is supposed to be carried out to optimize antimicrobial activity and to minimize toxicity and production costs.2 Strengths of AMPs2.1 Advantages in biophysical properties From the advances of peptides synthesis and structural-functional properties of AMPs, these polypeptides present great advantages and expose some aspects which need to be further investigated. With broad spectrum antimicrobial activities, high selectivity and potency, good efficacy, safety and tolerability, the antimicrobial peptide has shown increasing potential in global peptide drug market (Fosgerau and Hoffmann, 2015). 2.2 Advances in peptide synthesisThere have been considerable advances and strengths in aspects of AMPs especially in peptides synthesis. Employing solid-phase peptide synthesis(SPPS) method to repeat the addition of required amino acids for synthesis has become the major synthesis approach of peptides. Initially, the success of the Fmoc chemistry was contributed to that they could quickly prepare peptides suitable for antibody production using inexpensive machines and the process of Fmoc SPPS method was also easy to automate. The concern with the Boc technique has been conquered eventually through Fmoc SPPS mostly because of the improvements in purity of the Fmoc building blocks and the success of applying pseudoprolines and backbone protection to the synthesis of long peptides overcoming the difficult sequence problem. Though it is still far from meeting the potential of Fmoc SPSS, the constant improvement of side-chain protection strategies and increasing purity of the building blocks have made previously unobtainable, lengthy targets accessible, which was a prominent progress in peptide synthesis and made the production of antimicrobial peptide more effective and easier to acquire(Behrendt, White and Offer, 2016). 3 Growth potential of AMPsAntimicrobial peptides (AMPs), which have been discovered and isolated from organisms as diverse as plants, insects, amphibians, humans and even bacteria, are also called host defense peptides. AMPs are a diverse class of molecules that function as first line of defenses against microbial threats of many species. While antibiotics are losing effectiveness gradually in the current immunity system, AMPs are becoming a promising alternative, proposed as a potential source of novel anti-infectives (Wang, Li and Wang, 2008).To develop effective treatments for the infection of drug-resistant bacteria and other organisms, considerable effort has been made into identification, isolation, and activity testing in antimicrobial peptides. According to the relative clinical trials, natural AMPs, due to their diverse origins and evolution, target many microbial species and can exhibit functions in vitro activity, however, are low in vivo activity. The labile nature of peptides and potential toxicity concerns have prevented development of systemic applications and have hindered AMP clinical development. Recently, the new approach called for the design of synthetic sequences has been able to optimize sequence and overcome some of the limitations observed from natural antimicrobial peptides. Success of designed AMPs in vivo and in vitro activity data highlighted the rationality and potential of modified AMPs (Jonathan et al., 2014).Since cationic charge and significant proportion of hydrophobic residues may be two typical features for AMPs, and the common mode of action involves disrupting the integrity of the bacterial cytoplasmic membrane, the structural-activity relationship studies of AMPs require important status and research to demonstrate the designed details of the process of peptide synthesis (Nguyen, Haney and Vogel, 2011). For example, in the case of amphipathic α-helical AMPs, the different biophysical parameters, including the net charge, chain length, helicity, hydrophobicity and hydrophobic moment are responsible for their cell selectivity. The drug discovery was mainly contributed to the investigations helping identify the properties of the synthetic peptides, and these antimicrobial peptides became attractive drug candidates for the treatment of drug-resistant bacteria infection and inflammation because of their effective properties (Wang et al., 2010).Apart from the structural diversity, a common cationic antimicrobial peptide has another feature that it always has an amphipathic structure which allows it to bind to the membrane interface. The interaction of the peptides with membranes is an important requirement for most antimicrobial peptides. Though there are a wide range of peptides with different chemical structures exhibiting antimicrobial activities, some common properties, like cationicity and hydrophobicity, in some cases are shown to be important features which deserve to be focused on (Epand and Vogel, 1999).4 ConclusionThe wide range of natural AMPs in various species clearly indicate the ability to inactivate diverse bacterial species in various bioenvironments. Thus, future studies on AMPs should be focused on illustrating the critical structural determinants and mechanisms of activities of natural AMPs. More researches of the application of these structure-function relationships to the rational design of synthetic AMPs are supposed to be carried out to optimize antimicrobial activity and to minimize toxicity and production costs (Jonathan et al., 2014). ReferencesFosgerau, K. and Hoffmann, T. (2015). Peptide therapeutics: current status and future directions. Drug Discovery Today,20, 122-128.Behrendt, R., White, P. and Offer, J. (2016). Advances in Fmoc solid-phase peptide synthesis. Peptide Science, 22, 4-27.Wang, G., Li, X. and Wang, Z. (2008). APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Research,37, D933-D937.Nguyen, L., Haney, E. and Vogel, H. (2011). The expanding scope of antimicrobial peptide structures and their modes of action. Trends in Biotechnology, 29, 464-472.Wang, P., Nan, Y., Yang, S., Kang, S., Kim, Y., Park, I., Hahm, K. and Shin, S. (2010). Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich α-helical model antimicrobial peptide and its diastereomeric peptides. Peptides, 31, 1251-1261.Epand, R. and Vogel, H. (1999). Diversity of antimicrobial peptides and their mechanisms of action. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1462, 11-28.Jonathan D Steckbeck, Berthony Deslouches Ronald C Montelaro (2014) Antimicrobial peptides: new drugs for bad bugs?. Expert Opinion on Biological Therapy, 14, 11-14.
3. 设计方案和技术路线
First, solid-phase peptide synthesis (SPPS) method was employed to repeat the addition of required amino acids for synthesis from the oxime end (carboxyl group) to the oxime end (amino group). Fmoc was used to protect the side-chain of the amino acids participating in the reaction in case that side reactions occurred, and the C-terminal was released and activated before the reaction. The resin was added to immobilize the polypeptide - KWPLIGGILKKVL-NH2 and was removed in the end. The final product was lyophilized for use. Through comparative evaluation of mass spectrometry, HPLC, bioinformatics analysis, biological data, and known sequence information, the molecular weight of the polypeptide was determined, and the nature of the peptide was tentatively verified. Finally, functions of the peptide was identified and screened through relevant antibacterial experiments. The antibacterial effects of the polypeptide on Gram-negative E. coli, Gram-positive bacteria S. aureus, and fungi C. albicans were observed and their minimum inhibitory concentrations and the minimum bactericidal concentration were determined.Technical route: Review literature---Peptide Synthesis---Sequence identification--- Bioinformatics analysis--- Functional screening
4. 工作计划
TIME CONTENT PROGRESS RATE2022.03.03~03.08 Read articles regarding to the experiment and do the presentation completed2022.03.09~03.26 Do the experiment completed2022.03.27~04.04 Read relative articles and write the initiating report and the review paper completed2022.04.05~04.15 Finish the experiment completed2022.04.16~05.15 Analyze the experimental data and write the research paper To be completed
5. 难点与创新点
Solid-phase peptide synthesis (SPPS) based on Fmoc chemistry has become a commonly used technique recently, as it can be easily conducted using inexpensive automated machine and can prepare required peptides quickly, which is suitable for rapidly emerging field of peptide. The Fmoc SPPS technology provided a solution to the previous limiting condition of the Boc technique, as the deprotection conditions were compatible with modified peptides. The iterative use of TFA could cleave small amounts of the side-chain protecting groups at each cycle and cause progressive loss of peptide from the polymer support. In contrast, Fmoc SPPS provided an orthogonal combination of temporary and permanent protecting groups.
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