Comparative Study Of Biological Activity Of D-/L-Enantiomeric Antimicrobial Peptides

There are two major obstacles that inhibit the use of antimicrobial peptides as novel anti-infection therapeutics: first, the biological activity of antimicrobial peptide is decreased severely under conditions of physiological salt concentration or human serum; second, antimicrobial peptide is digested in vivo by varying proteases. Utilizing antimicrobial peptide V13K as a template, we studied biological activity of antimicrobial peptides under physiological situations.In this study, L-V13K and its enantiomeric isomers D-V13K were synthesized to compare biophysical and biologic properties within physiological and non-physiological conditions. In biophysical part, peptide hydrophobicity was determined by RP-HPLC; second structure of peptides in different solutions was determined by circular dichroism spectroscopy. Biological activities including antimicrobial activity of peptides and hemolytic activity were determined; antibacterial mechanism of action in vivo was discussed based on NPN uptake experiment, proteolytic stability assay, etc.Antimicrobial peptides D/L-V13K were successful synthesized by Fmoc solid phase peptide synthesis, samples were purified by preparative RP-HPLC technology, the purity of samples was confirmed more than 95% by analytical RP-HPLC. The correct products of D/L-V13K were further identified by amino acid analysis and mass spectrometry.In this study, D/L-V13K exhibited antimicrobial activities against representative strains of Gram-negative and Gram-positive bacteria. D-V13K was resistant to the degradation of trypsin and pepsin, but L-V13K was gradually hydrolyzed by two proteases, which resulting in the differences of biological activity of D- and L-antimicrobial peptides, this also indicates that D-V13K has certain advantages when applied in vivo. In the non-physiological conditions, D/L-V13K demonstrated the same or similar antimicrobial activities (within experimental error) against Gram-negative bacteria and some Gram-positive bacteria. Both peptides exhibited the same negligible toxicity (500μg/ml) against human erythrocytes. D/L-V13K, shown by circular dichroism, presented nearly symmetrical mirror images ofα-helix structure in hydrophobic environment. All these indicated that the mode of action of D/L-V13K on the membranes of bacteria or erythrocytes may be similar; both peptides were directly interacting with the cell membrane without specific receptors with chiral structure or enzymes of membrane. For various Gram-positive bacteria, the bactericidal activity of antimicrobial peptides V13K varied greatly, this may be related to the environment of cell wall surface of Gram-positive bacteria. For the same Gram-positive strain, antibacterial activity of D-V13K and L-V13K was quite different, D-V13K had higher activity than L-enantiomer. Such as for Bacillus subtilis, this bacterium secreted an extracellular protease that can degrade L-V13L, which causing the different activity between D- and L- peptides. Surprisingly, D-V13K had the high activity (16μg/ml) against Staphylococcus aureus (include MRSA) which is a great threat as superbug to human health now. D/L-V13K antibacterial activity was weakened under physiological conditions (adding Na+ , Ca2+ or human serum albumin). Under physiological salt concentration, salt ions decreased antibacterial activity which may be attributed to the weakening of electrostatic interactions between positively charged peptides and negatively charged cell membranes; another reason may be that the stability ofα-helical structure was disrupted under high salt conditions, which is very important for antibacterial activity of D/L-V13K. With human serum albumin in the solution, antimicrobial peptides may combine with albumin. There may be a dynamic equilibrium between free and protein bound peptide molecules, this effect decreased the antibacterial activity of antimicrobial peptide V13K. For Gram-negative bacteria, D/L-V13K were resistent to the effect of physiological concentration of Na+; in contrast, D/L-V13K were very sensitive to the Ca2+, which can reduce the activity of two peptides to the same degree. For Gram-positive bacteria, the antagonism of Na+ was relatively larger for the antibacterial activity of peptide D/L-V13K, it is significantly larger than the antagonism of Ca2+. In the presence of human serum albumin, antibacterial activity of D/L-V13K was reduced to lager extent (except Staphylococcus epidermidis), this has hampered antimicrobial peptides D/L-V13K to be developed as safe and effective systemic drugs. NPN uptake experiments of Pseudomonas aeruginosa illustrated that D/L-V13K enantiomers could realize the purpose of sterilization by penetrating the outer membrane of Gram-negative bacteria. NPN fluorescence intensity is related with the antimicrobial peptide concentration within a certain range. The rate of NPN uptake directly reflects the interaction of antimicrobial peptide D/L-V13K with outer membranes. In the physiological salt concentration, NPN fluorescence was decreased after the addition of Na+ and Ca2+, antimicrobial peptides D/L-V13K promoted NPN uptake was completely inhibited by the divalent cations Ca2+. In the presence of Ca2+, fluorescence intensity decreased to the level similar to the negative control, which is fully consistent with antibacterial experimental results.Through comparison of D/L-V13K biological activity in different influencial factors, we conclude that D-V13K has stronger antibacterial activity, especially against Gram-positive bacteria S. epidermidis, and could become a drug candidate with high potential.Effects of physiological conditions for antimicrobial peptide V13K is larger than some other antimicrobial peptides with relatively high activity under physiological conditions. Activity of antimicrobial peptide V13K against Gram-negative bacteria is affected by Na+ at least, peptides are resistent to Na+ in certain extent, and we need more data to study physiological conditions effects on antibacterial activity of antimicrobial peptides. It is urgent to find the way that antimicrobial peptides overcome the inhibitory effects of physiological conditions (salts or serum protein). Through our study, although the physiological conditions may weaken biological activity of amphipathicα-helical antimicrobial peptides alike V13K, V13K is still expected to be developed as a highly effective antimicrobial agents.