Unnatural Amino Acids with Abbreviations
1.3. Effect of Environmental Factors on the Activity of AMPs 1. Salt
Figure 1.16. Primary sequence of some representative examples of anionic AMPs.
1.3. Effect of Environmental Factors on the Activity of AMPs
hydrogen bonds in between the CO and NH groups of successive turns. The H-bond interactions are dependent on the salt concentration and the pH of the medium. Presence of salts not only affect the structure but also the peptide-lipid interactions. The rate of interaction of AMP magainin with the bacterial membrane mimic bilayers was shown to be slower in the presence of salts.266,267 Ghosh et. al. proved through MD simulations that the initial rate of interaction in between the AMP and the membrane mimetics (SDS micelles) was delayed in the presence of salt.267 In a separate study, they also established that the dissociated AMP and membrane mimetic systems were selectively more stabilized in the presence of salt, which made the dissociation of AMP: membrane mimetic complex faster in the presence of salts (unpublished results).
Several strategies have been applied to generate synthetic salt tolerant peptides both from natural AMPs as well as de novo designed peptides (Figure 1.17). One of the strategies is to modify the natural AMP sequences. For example, truncated 28-mer analog of Dermaseptin S4 was found to have salt tolerant against E. coli at high salt concentrations while the 14-mer peptide was inactive.268 Hybrid peptides designed from combining the insect cecropin and bee mellitin were found to be active in high salt concentrations.269 Meitzner and coworkers developed de novo designed peptides rich in Val and Arg which were salt tolerent in their activity towards P. aeruginosa and S. aureus.111,270 Peptidomimetic approaches, like use of D- amino acid residues271 and bulky unnatural amino acid residues272 are also reported to improve the salt tolerance of antimicrobial activity.273,274 Macrocyclization and introduction of di- sulphide bonds were reported as a way of introducing salt tolerance in synthetic peptides.263,275 Yang and coworkers produced constrained analog of tachyplesin which was active at high salt concentrations.263 Dimerization of AMPs via the disulphide bond formation or through side chain of Lys was shown to be an effective way to induce salt tolerance of antimicrobial activity.276,277 As the loss of helical content in presence of salt was considered as a plausible
reason for the loss of activity of the AMPs, introduction of helix stabilizing sequences at the C- and N- termini of the AMPs led to the retention of activity in the presence of high concentrations of salt.278 Though there are several modifications reported to develop salt sensitivity in the synthetic peptides, unfortunately most of them are peptide or microorganism specific. To develop more general methods to develop salt tolerance, studies are required in greater details to understand the mechanism of inactivation in order to come up with robust design principles.
Figure 1.17. Various strategies adopted for generating salt tolerant synthetic AMPs.
1.3.2. pH:
Activity of the AMPs is dependent on the pH of the environment279 as the pH determines its physicochemical properties and hence its mode of action. The potency of the AMP depends on its therapeutic site as most of the times the local pH may vary from the physiological pH (pH
7.4). For example, in the local sites for inflammation like the abscess, the pH is much acidic
owing to the local increase in the concentration of lactic acid and fatty acid by-products formed from the bacterial metabolism.280,281 In skin the acidic environment (pH = 4-6) inhibits bacterial growth and promotes wound healing.282,283 pH affects the physicochemical properties of the AMPs i.e., the protonation state of the side chains of charged amino acid residues leading to a change in the overall charge of the peptide. Moreover, pH might also effect the secondary structure of the AMPs, thus varying their activity in the process. Additionally, pH might also vary the nature of the bacterial surface, thereby affecting the AMP membrane binding.284 It has been established that the activity of AMPs containing His residue increases at lower pH while it diminishes at the higher pH. At a lower pH, the His residues are mostly protonated (pKa = 6.5), thereby increasing the overall charge of the peptide. On the other hand, at the higher pH, His deprotonates reducing the overall charge of the AMPs and making them less potent against anionic microbial membranes.262,279,285,286 pH also influences the activity of the anionic AMPs by increasing the overall positive charge at a lower pH.287,288 Wiey and coworkers established that the activity of AMPs reduced against Gram-negative bacteria in general as opposed to an increase in the activity against Gram-positive bacteria with increasing pH.289 This was attributed to the change in the charged state of the molecules present in peptidoglycan layer in the Gram-positive bacterial strains at higher pH, such that they did not interfere with the diffusion of the AMPs through the peptidoglycan layer to interact with the inner membrane. Welsh and coworkers demonstrated a decrease in the antimicrobial activity of -defensin and LL-37 in the acidic pH against S. aureus and P. aeruginosa and a decrease in the synergistic effect seen in between the two at an acidic pH as well.289 Alternatively both the peptides showed enhanced activity against S. aureus at a basic pH where a reduction in the overall charge might facilitate insertion through the membrane.290
1.3.3. Proteases:
One of the major drawbacks of the AMPs in being used as therapeutic molecules to combat bacterial infection, lies in their small half-life. AMPs constituted of all amino acid residues are susceptible to cleavage by the proteases. Proteases are present in the blood serum and AMPs administered orally or intravenously encounter them. Degradation of the AMPs in the blood circulation even before reaching their therapeutic target reduces their efficiency. Incorporation of D-amino acid residues,291-293 unnatural analogs of amino acid residues,294 N and C terminal protection,117,295,296 lipidation,297,298 and cyclization299 are some of the strategies adopted for conferring protease resistance to synthetic AMPs (Figure 1.18).300
Figure 1.18. Various strategies adopted for generation of protease resistant synthetic peptide.300