Virtual Composite Design Of Small Molecule Antimicrobial Peptides, The Screening Effect

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.