Peptide design and synthesis
In the present study, the peptides KWKLFKKI-CONH2 (P2) and KWKLWKKI-CONH2 (P3) were prepared from Cecropin-A family of KWKLFKKIQIAK-CONH2 (P1) using the Rink amide resin support and the Fmoc- chemistry solid phase peptide synthesis.23 Cecropin-A is a 36-residue long peptide with a high intrinsic helical propensity. It contains 8 cationic sites which are critical for bacterial cell selective binding. The aliphatic index24of cecropin-A is 108.11. We envisioned that a short peptide comprising the N-terminal and C-terminal segments of cecropin-A would elicit antimicrobial activity as it would have 6 cationic sites and an aliphatic index value of 105.83. The predicted intrinsic helix propensity25,26 of the peptide should enable it to adopt α-helical conformation in a hydrophobic environment and thereby promote membrane permeabilization. The six cationic sites would favour strong ionic interactions between the positively charged sites on the peptide and the negatively charged sites on bacterial membrane and thereby enhance cell selective activity. Accordingly, the peptide sequence of P1 was spliced out from the framework of cecropin-A.
We used peptide P2 and P3 as a framework to systematically alter peptide hydrophobicity by (i) deleting the C-terminus of the peptide (minimalist design approach) and (ii) substituting a D-amino acid derivative, α-(2,5,7-tri-tertiarybutylindol-3-yl)alanine in place of L-phenylalanine residue to increase the overall hydrophobicity and abolish secondary structural propensity.27-30 Therefore, incorporation of another residue of α-(2,5,7-tri-tertiarybutylindol-3-yl)alanine into the sequence of P2 would result in a peptide P3 with very high hydrophobicity while retaining the cationic nature of P2. Thus, P3 can be expected to be the most potent among the short peptides derived from cecropin A. The peptide sequences are shown in Table 1. The schematic line structure of peptides given in Figure 1 (Scheme 1 and 2).Observation of molecular mass is critical to establish the structural integrity of the peptides. Mass spectrometer also provides information related to fragment ions, the ability to form adducts with ligand and metal ions as well as peptide aggregation (dimer, trimer etc.). The ESI mass of spectra of peptides P1, P2 and P3 are presented in Figure S3-S5. The calculated mass using the above method corroborates with the monoisotopic mass of P2 (m/z = 1256.9) within the instrument acceptance limit of unit mass resolution. The calculated mass using the above method corroborates with the monoisotopic mass of P3 (m/z = 1464.1) within the instrument acceptance limit of unit mass resolution.
Table 1 Amino acid sequence of ultra short peptides related to Cecropin-A
Name
|
Sequence
|
Aliphatic Content
|
Positive Charge
|
Molecular Weight (m/z)
|
HPLC Purity
(%)
|
P1
|
KWKLFKKIQIAK-CONH2
|
46
|
6
|
1529.9
|
90
|
P2
|
KWKLFKKI-CONH2
|
35
|
5
|
1256.9
|
90
|
P3
|
KWKLWKKI-CONH2
|
44
|
5
|
1464.1
|
88
|
W = α-(2,5 7-tri-tertiarybutylindol-3-yl)alanine
Peptide homogeneity and integrity
The preparative (crude peptide) and the analytical (purified peptide) reversed phase HPLC chromatograms of the peptides are shown in Figure 2 (Figure S1 and S2). Impurities are frequently present in multi-step synthesises. By using solid-phase step-wise synthesis techniques, the amount of impurities can be greatly reduced. The benefit of solid-phase synthesis is attested to by the HPLC chromatogram of the crude peptides in Figures 2. The peptides P2 and P3 were purified to homogeneity.
To determine whether the peptides' structural integrity is intact, molecular mass observation is essential. The ability of fragment ions to form adducts with ligand and metal ions, as well as peptide aggregation (dimer, trimer, etc.), are also revealed by mass spectrometry. The ESI mass of spectra of peptides P1, P2 and P3 are presented in Figure S3- S5. Since the ESI source in mass spectrometry has the ability to produce the multiple charged ions, P2 and P3 might also produce multiply charged species. The calculated mass using the above method corroborates with the monoisotopic mass of the peptides was calculated using Somion method31within the instrument acceptance limit of unit mass resolution. The absence of mass peaks corresponding to any other impurity and side chain modified forms of the peptide showed the integrity of the peptides.
Peptide hydrophobicity
The retention patterns of peptides after interaction with the hydrophobic environment of the column matrix during reversed phase HPLC are a particularly good approach to depict apparent peptide hydrophobicity, and the retention periods of peptides are very sensitive to this conformational condition. An amphipathic -helical peptide's non-polar face is its preferred domain for attaching to a reverse phase column's hydrophobic matrix. According to RP-HPLC retention time, the observed hydrophobicity of the peptides in this investigation is in the following order: P1 ≤ P2 < P3 (Table 2). In the case of P2 peptide, the deletion of four C-terminal amino acids leads to a reduction in hydrophilicity32 [net hydrophobicity value for QIAK = - 0.350 (Eisenberg, 1984)] which is manifested by the slight increase in the retention time of P2 as compared with P1. Incorporation of one more α-(2,5,7-Tri-tertiary butylindol-3-yl)alanine in place of phenyl alanine increases the hydrophobicity to a greater extent. Two α-(2,5,7-Tri-tert-butylindol-3-yl)alanine residue substituted peptide P3 showed the highest hydrophobicity among the peptide analogs (tR, 35.23 min.; Table 2). As shown in Table 1, the number of the hydrophobic interactions with the peptides correlates with the observed hydrophobicity values of the peptides with the same amino acid composition. The HPLC trace of P1, P2 and P3 is also given Figure 3 for comparison. The peptide P1 has 12 amino acids. The C-terminal tetra peptide segment contributes a hydrophobicity value of -0.350 to P1. The net hydrophobicity value of the C-terminal sequence QAIK was calculated from the normalized consensus hydrophobicity values of different amino acids.24 Accordingly, the peptide P2 which lacks the C-terminal segment in P1 can be expected to be more hydrophobic than the latter.24, 26, 33
Table 2: Biophysical data of peptides related to Cecropin-A
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)
aThe hydrophobicity order of peptides was deduced from observed tRvalues on a RP-HPLC at room temperature and at pH 2.
Change in hydrophobicity = tR(X –P1), difference in retention times between peptide analogs and the native peptide, P1.
bThe mean [θ]222 values (in deg . cm2 . dmol-1) at wavelength 222 nm were measured at RT in buffer (10 mM PO42-, pH 7.4), in buffer containing 80% TFE and in SDS micelles using circular dichroism spectropolarimeter.
cThe helical content (helix percentage) of a peptide relative to the molar ellipticity value of peptide P2/ P3 in buffer (10 mM PO42-, pH 7.4), in buffer containing 80% TFE and in SDS micelles.
From a closer look at the hydrophobicity order seen in Figure 3 and the binding behaviour of P1, P2 and P3 to zwitterionic POPC vesicles; it is conceivable that the extent of peptide binding to lipid membrane directly correlates with the hydrophobicity.34 Even though, P1 is a longer peptide and has an additional positive charge, it fails to bind POPC vesicles due to its reduced hydrophobicity. The binding behaviour of these peptides against anionic POPC-POPG (7:3) vesicles also follows the same trend (P1<P2<P3). Taken together, binding to both acidic and zwitterionic lipid membranes appears to be dominated by hydrophobicity of the peptides
Reduction in length and incorporation of D-amino acid derivative abolishes helix forming ability of peptides
CD spectra of the peptides in PBS solution (to mimic physiological conditions), TFE (for mimicking hydrophobic environment) and 30 mM SDS micelles (to mimic the lipid membrane) were recorded to study the environment dependant conformational transitions which would have a bearing on the activity spectrum of the peptides. Peptides P2 and P3 did not show any significant signal representing either turn or helical structure in PBS buffer pH 7.4 as seen in Figure 4 and Table 2. However, the negative minima at ~222 nm in the CD spectra of peptides in TFE suggest the induction of α-helix secondary structures in the non-polar environment.35-37Similarly, the CD spectra of peptides in 30 mM SDS that mimics the lipid membranes also show the induction of helical structure. The random-to-helix transition of peptides in membrane environment is critical for eliciting their activity as the helix provides amphipathicity to the peptide. When the charges and the hydrophobic moieties are segregated along the helix, the peptide makes a tight binding with the bacterial membrane and then inserts into the membrane and causes membrane permeabilization.
Spectra recorded in neat TFE are generally used to determine the relative helix content and thereby the amphipathicity of the peptides. As seen clearly in Figure 4, the peptide P1 displays the moderate helix content. It is evident from the previous chapter and other studies38 that the incorporation of D-amino acid α-(2,5,7-tri-tert-butylindol-3-yl)alanine in the place of L-tryptophan clearly reduces the helix content. This observation is further confirmed with P2 which lacks the four C-terminal residues. Shortening the peptide length has also been shown to reduce the helix propensity of the peptides.
It order to confirm that increasing hydrophobicity alone is not sufficient to increase the α-helical structure of the peptides in non-polar as well as helix promoting environments, the CD spectra of P3 was recorded in aqueous buffer, neat TFE and 30 mM SDS . As shown in Figure 4, some degree of ordering in the conformational freedom of P2 and P3 appears to set in when these peptides are placed in non-polar and membrane mimicking environment. However, there is a clear preference for random conformation presumably due to the incorporation of D-amino acid compounded with the short length of the peptides. It is imperative to note that non-helical antimicrobial peptides are better candidates than helical antimicrobial peptides for therapeutic purposes as they do not have the ability to form membrane pores, unlike helical peptides, which causes the leakage of cellular contents in the near vicinity.
Membrane binding assay using intrinsic tryptophan fluorescence
Steady state fluorescence spectroscopy is routinely used to determine the binding affinity of peptides to lipid membranes and other substrates. The intrinsic fluorescence of the tryptophan moiety was exploited to explore the interactions between, P2 and P3 and lipid vesicles made of POPC and POPC/POPG (7:3). The enhancement in the steady state fluorescence intensity of peptides as a function of lipid concentration is presented in Figure 7. As it is clear from Figure 6 the POPC vesicle induced enhancement in the fluorescence intensity of P2 is not significant as compared with that observed with POPC/POPG (7:3) lipid vesicles, suggesting the anionic lipid selective binding and interaction of P2. A similar behaviour is not observed with P3 (Figure 7). It is imperative to note that, P3 interacts with both POPC and POPC/POPG (7:3) vesicles. Certainly, further alkylation in the case of P3 peptide results in enhanced binding to both anionic and zwitterionic lipid vesicles, and therefore may not be suitable for cell selective interactions and targeting. Peptide P2 appears to hold the promise as it interacts with only anionic POPC/POPG (7:3) vesicles at a low peptide/lipid ratio. While the POPC vesicle induced changes in the steady state fluorescence of P1 is negligible (Figure 5), the quantum of fluorescence observed at low peptide/POPC/POPG (7:3) ratio is remarkably high and comparable to that observed with P1.It is clear from Figures 5-7 and, that P2 is as selective as P1 and the most active of the three peptides designed for this study.
A peptide antibiotic must have the ability to bind the cell wall of bacterium and then insert itself into the membrane to alter the permeability characteristics of the membrane. The blue shift observed in the fluorescence emission maxima of P2 and P3 upon binding to lipid vesicles (Figures 6B and 7B) suggested that the peptides were indeed in the membrane inserted state. Such blue shifts were not observed from P2 when POPC vesicles were added suggesting a non-lytic membrane embedded state of the peptide. The increase in the fluorescence intensity of P3 upon addition of POPC vesicles suggests binding to lipid vesicles presumably facilitated by the introduction of second D-amino acid α-(2,5,7-tri-tert-butylindol-3-yl) alanine in the place of L-phenyl alanine of the peptide. The observed blue shift indicates that the peptide P3 is inserted into even the zwitterionic membrane implying the need for the hydrophobic-hydrophilic balance in designing peptides for membrane selective targeting and delivery. The peptide: lipid molar ratio at equilibrium binding to POPC/POPG (7:3) vesicles were obtained from the plot of bound-peptide versus amount of lipid (initial slope of the curves) and presented in Table 3.
Table 3: Binding affinity for POPC-POPG (7:3) vesicles
Peptide
|
Peptide : Lipid molar ratio at equilibrium binding to POPC/POPG vesicles
|
P2
|
1:53
|
P3
|
1:06
|
P1
|
1.34
|
Antimicrobial activity
Antimicrobial peptides can be grouped into hemolytic and non-hemolytic. The non-hemolytic AMPs are either cyclic short peptides or short linear peptides with moderate hydrophobicity.39, 40 The hemolytic or cell lytic AMPs are usually longer in size (more than 20 amino acids) and highly hydrophobic. In 8-residue peptides such as the ones used in this study, increasing the hydrophobicity is limited. Incorporation of even one modified tryptophan moiety greatly increases the hydrophobicity of the peptide to an extent that the peptide loses the selective affinity for anionic membrane and binds to both zwitterionic and anionic lipids. Subsequently, the hydrophobic peptides show a lower MIC value against bacteria and kill mammalian cells as well. It’s intriguing that the synthetic peptides P2 and P3 both exhibit strong antibacterial and mild hemolytic action. Similar behaviour has been noted for various substances that exhibit anticancer and antibacterial properties because cancer and bacterial cell membranes include acidic lipids.39 P2 and P3 might consequently be able to kill cancer cells just as they can kill germs.
Presented in Figure 8 is the graph of antimicrobial activity elicited by P2 and P3 against both Gram positive and Gram-negative bacterial strains. Antimicrobial activity is observed in the concentration range from 5-80 µg/mL. The peptide P2 and P3 are less cationic than P1. It is interesting to note that, while peptide P1 shows activity from 20-60 µg/mL, P2 and P3 that have five cationic sites display activities at much lower concentrations (10-50 µg/mL). For S. aureus (15 µg/mL), E. coli (10 µg/mL), P. aeruginosa (45 µg/mL), and B. Subtilis (10 µg/mL), peptides P3 have lower MIC in comparison to the parent peptide P1 and P2. Likewise, Peptide P2 has also displayed higher activity than parent peptide P1.The peptide P3 has demonstrated the highest antibacterial activity, which may be because its five cationic sites exhibit actions at much lower doses that are comparable to the medicinal medication amikacin, which is commercially available (10-15 µg/mL). This finding show in this case as several peptides are hemolytic with less than five cationic residues is required to produce antibacterial action. The decrease in P3 MIC values and the peptide's increased hydrophobicity are correlated.
Hemolytic activity
Hemolysis assay21, 22 is the chief method to assess the toxicity of any drug candidate. Since red blood cells are highly sensitive to chemical agents like salts, detergents, polymers, pH, temperature, etc., hemolytic ability of a molecule is often assessed before considering any molecule as a pro-drug. A comparison of the relative potencies of the peptides, P1, P2 and P3 are presented in Figures 9A-B. The peptide P2, the most hydrophobic peptide considered for this study is highly toxic as it releases the cellular contents of red blood cells even at very low concentrations (~92 % hemolysis observed with 20 µg/mL peptide). Whereas the peptide P2 containing only one modified tryptophan residue, does not show any significant amount of lysis at MIC. However about 50% hemolysis was observed with 100 µg peptide. Peptide P1 is clearly non-hemolytic in spite of its longer length (12 residues) and six cationic residues. Secondary structure of the peptides does not seem to have any role in eliciting hemolytic activity as all two peptides are in extended conformations.
Peptides P2 and P3 are shorter and hydrophobic than P1. A direct comparison between P1 and P2 threw some light with regard to the often quoted “hydrophobic-hydrophilic balance” for antimicrobial activity. While P3 is the most hydrophobic peptide used in this study that inserts into even zwitterionic membrane (POPC vesicles), P2 binds to only anionic POPC/POPG (7:3) vesicles (see fluorescence data). This implies a limit to peptide hydrophobicity in order to selectively target and permeabilize the lipid membranes.
Peptide P2 is shorter by four residues and less cationic than P1, yet it has comparable, if not, slightly more hydrophobicity. While the presence of an additional positive charge (lysine residue) in P1 does not seem to enhance the antimicrobial activity, it clearly reduces the overall hydrophobicity of P1 (See Figure 3 and Table 3). It appears that one charged amino group in the side chain of a peptide can nullify the hydrophobic influence of 11 methylene units present in the side chains of a peptide.
Peptide P2 differs from P1 in that it lacks the C-terminal tetra peptide sequence QIAK present in P1. Loss of QIAK makes P2 shorter and contributes to a slight increase in hydrophobicity. Like peptide P1and P2 displays membrane selective interactions, albeit with enhanced affinity. This enhanced affinity for anionic membranes despite the loss of a positive charge could only be explained by the hydrophobic cluster present in P1 (i.e., presence of two modified tryptophan residues separated by only. The smaller sizes of P1 and P2 appear to favour clustering of hydrophobic and cationic moieties. Even though P1 cationic charges and the required hydrophobicity, clustering of hydrophobic and cationic moieties in P2 appears to provide the leverage for the enhanced antimicrobial activity.
The octapeptides designed for the current study exhibit antimicrobial activities that are similar to those described earlier, which are made up of L-amino acids only. The selective activities observed for P1 and P2 as compared with the activities of the highly hydrophobic P3 suggests that hydrogen bonding or electrostatic interactions through the protonated amino groups are involved in the antimicrobial activity. The higher level of hemolytic activity observed for P3 may be due to the hydrophobicity of the modified tryptophan moiety. Due to their short length, octapeptides are predicted to display their activities through preventing cell growth and development rather than by lysing the cells. For longer antimicrobial peptides, such as magainins40 and cecropins41, spanning the lipid bilayer of a membrane and creating a channel through the membranes has been proposed. This requires a minimum of 21 residues in the helix form.42 These short peptides may bind to the lipopolysaccharides of the bacterial cell membranes in a way that is similar to that of the magainins42 because of their cationic nature, increasing cell permeability. This is corroborated by the fact that, in comparison to Gram-negative bacteria, which have less complex cell walls and no outer membrane, Gram-positive bacteria exhibit higher levels of activity. The studies described above indicate that new potent antimicrobial compounds can be designed based on the biophysical properties of natural and unnatural amino acids to suit the therapeutic requirements in the future.43