A novel short N-terminal cationic and hydrophobic peptides, KWKLFKKI-CONH2 (P2) and KWLWKKI-CONH2 (P3) are a class of Cecropin-A family of KWKLFKKIQIAK-CONH2 (P1) was designed using Fmoc-chemistry solid phase peptide synthesis protocol, where W stands for α-(2,5,7-tri-tert-butylindol-3-yl) alanine residue. By comparing High performance liquid chromatograms or Mass spectrometry (LCMS or analytical HPLC), the purity, integrity, and homogeneity of the peptide were determined. The circular dichroism spectroscopy (CD) demonstrates that to detect conformational alterations during membrane contact, P2 adopts an extended structure in both polar and non-polar settings, as expected. Because of the presence of tryptophan derivatives, P3 occurs in an extended conformation. Peptide P2 exhibited an exceptional affinity for both zwitterionic POPC lipid bilayer and anionic POPC/POPG lipid bilayer membranes, whereas P3 preferentially interacts with POPC/POPG anionic bilayer rather than zwitterionic POPC lipid bilayer. Surprisingly, both peptides have good antibacterial activity against Gram-positive and Gram-negative microorganisms. It is important to note that the most hydrophobic P3 had more effectiveness against all test organisms than P2 and the control peptide P1. The toxicity of these peptides was examined using a hemolytic assay, and the results reveal that P2 and P3 have very little to no toxicity, which is important for P2 and P3 to be utilised as possible therapeutic agents. Peptides P2 and P3 were both non-hemolytic and appeared to be more capable due to their broad antibacterial activity.
Research Article
Design and Synthesis of N-terminal segment Peptides: A New Innovative Finding for Antimicrobial Activity
https://doi.org/10.21203/rs.3.rs-3457760/v1
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Cecropin-A
Non-hemolytic peptides
Steady-State fluorescence spectroscopy
Peptide-lipid binding interaction
Antimicrobial peptides
In the drug development perspective, it is vital that most of the attractive properties of
cationic antimicrobial peptides (CAPs) are still contained in the series of extremely short synthetic antimicrobial peptides. Even though these molecules contain amide bonds, they can hardly get consideration as antimicrobial peptides in the classical sense. Thus, synthetic antimicrobial peptidomimetics (SAMPs) have been proposed as a term to describe this class of compounds.1 In addition to Gram-negative bacteria, several Gram-positive bacteria have also been found to be highly active against SAMPs.2 Derivatives of these compounds show high selectivity for Gram-positive and Gram-negative bacteria when compared to red blood cells and satisfy the minimal structural requirements for cationic antimicrobial peptides. SAMPs have been found to share many of the appealing properties of cationic antimicrobial peptides, such as the ability of a representative SAMP to insert into the bilayers of large unilamellar vesicles, permeabilize both the outer and cytoplasmic membranes of Escherichia coli, and kill Staphylococcus aureus in an extremely rapid manner.2–5 While antimicrobial peptides are susceptible to proteolytic degradation, SAMPs have been found to have remarkable in vitro stability in human blood plasma.6 These compounds are potential prospects for new antibacterial therapies because to their potent antibacterial action against methicillin-resistant staphylococci, moderate toxicity towards human erythrocytes, and relatively high resistance to proteolytic enzymes.
The widespread use of antibiotics has resulted in the development of a large number of antibiotic-resistant strains. For example, MRSA has acquired resistance to antibiotics such as the fluoroquinolones7. The emergence of vancomycin-resistant Enterococcus strains has raised the fear as this resistance is likely to be passed to MRSA8. The necessity for novel antibiotics that are effective against the surfacing resistant bacteria is highlighted by these recent advances. It takes a lot of time and effort to screen a lot of novel compounds for the rational creation of new antimicrobial drugs. Antimicrobial drugs can now be found in just a few weeks thanks to the development of soluble combinatory peptide and organic chemical libraries. The identification of a number of hexapeptides composed of L-amino acids which exhibit antimicrobial activities against S. aureus.9–11 and E. Coli.12 in the concentration range from 3.0–20 µg/mL has encouraged the synthesis and development of very short peptides and peptide mimics as antimicrobial agents. Moreover, the activities of hexapeptides are greater or equivalent to those found for naturally occurring peptides (15 residues for the gramicidin, 23 for the magainins, 35 to 37 for the cecropins, and 30 to 43 for the defensins).13 Considering the high cost involved in the synthesis of long peptides, attention has been focussed on designing short bioactive peptides and their mimics in the recent times. Cecropin-A is one of the most studied antimicrobial peptides. It shows excellent activity against a wide spectrum of both Gram-positive and Gram-negative bacteria. However, cecropin-A is toxic to human cells at higher concentrations. The alpha helical structure and the lysine residues of cecropin-A have been attributed to its activity against bacteria.14 Very short peptides (6–12) residues have been identified and reported to be active against bacteria. Some of them even show broad spectrum activity.15 Moreover, many of these short peptides lack a stable helix structure, adapting mainly an unordered conformation under physiological conditions.
To expand the chemical diversity and to increase the resistivity of antimicrobial peptides against the enzymatic degradation in in-vivo systems, D- and unnatural amino acids have been incorporated.16–18 Using synthetic peptide combinatorial library (SPCL) approach, a library of peptides composed of all possible hexameric combinations of 18 of the 20 natural L-amino acids has been used to synthesize individual peptides, which were then tested for activity.19 Because most newly produced antimicrobial peptides are exclusively made of L-amino acids, they are susceptible to proteolytic breakdown and rarely exhibit action for systemic application. Antimicrobial peptides containing D- and/or unnatural amino acids in their sequences are believed to have higher stabilities towards proteolysis, increasing their duration of activity and applicability.
In this present study, the structure and activity profiles of three short peptides (P1, P2 and P3) are presented. We have extended our search for new antimicrobial agents by determining the activities of two new 8-residue peptide mimics made up of L- and D- amino acids against laboratory test organisms. All two peptides/mimics contain α-(2,5,7-tri-tert-butylindol-3-yl)alanine residue and exhibit cell lytic activity. The effect of incorporation of one or two α-(2,5,7-tri-tert-butylindol-3-yl)alanine residues on the structure as well as their binding affinities has been determined. The hemolytic and antimicrobial activities of these peptides were also examined. The retention times of these peptides are proportional to their aliphatic content. The role of hydrophobicity in peptide membrane selective attachment is discussed. A possible structure-activity connection resulting from peptide CD analyses and their biological activities are also discussed.
Novabiochem, Merck, Germany provided the Fmoc-Rink amide MBHA resin and the Fmoc-protected amino acids. From Avanti Polar Lipids Inc. in Alabaster, lipids were bought. Every other chemical used for this study came from Merck and was utilised exactly as it was delivered. Rink amide-methylbenzhydrylaminehydrochloride salt (MBHA) resin, 9-fluorenyl-methoxycarbonyl (Fmoc) protected amino acids (Merck India Pvt. Ltd), diisopropyl ethylamine (DIPEA), 1-hydroxy benzotriazole (HOBT), trifluoroacetic acid (TFA), thioanisole, and ethanedithiol, deutrated SDS, Pyridine, Silica gel (100 mess) and the peptide synthesis vessel were purchased from Sigma Aldrich - Merck Chemicals Pvt. Ltd (Bangalore, India). The bacteria were produced by Sigma Aldrich Chemicals Pvt. Ltd. (Bangalore, India), including Gram-positive Staphylococcus aureus (ATCC 9144) and Bacillus subtilis and Gram-negative Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 1688). Avanti Polar Lipids (Alabaster, AL) supplied the lipids employed in the study, such as 2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine (POPC) and 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)sodium salt (POPG). The following solvents were purchased from Spectrochem Pvt. Ltd. (Mumbai, India): acetonitrile, water, dichloromethane, hexane, ethyl acetate dimethylformamide, chloroform, and methanol. Water from the NANO pure A filtration system was used to create buffers. No additional purification was performed before using any of the chemicals.
Preparation of α-(2, 5, 7-tri-tert-butylindol-3-yl)alanine
D-Tryptophan (4.79 g) was reacted with t-butanol (24 mL) and TFA (73 mL) and the contents were stirred at room temperature for 72 hours (Scheme 1). Then the reaction mixture was distilled off under vacuum using Buchi rotavapor flask, to get the black oily material. To this black oil, potassium hydrogen carbonate and water were added, until the solution became neutral. Care had been taken to arrest the effervescence. During the addition of base, a pinkish gum-like substance was created by repeatedly triturating the oily material with a spatula. A gum-like substance was crystallised from a mixture of 50% ethanol and water after the aqueous layer was decanted. The first batch of crystalline material was recrystallized from 50% ethanol in water to produce an amorphous crystalline solid of - (2,5,7-tri-tert-butylindol-3-yl) alanine.
Preparation of Fmoc α-(2,5,7-tri-tert-butylindol-3-yl)alanine
A weighed quantity (1.85 g) of α-(2,5,7-tri-tert-butylindol-3-yl)alanine was mixed with 8 mL pyridine and the contents were stirred at 0°C for 15 min. using the magnetic stirrer. Fmoc chloride (1.55 g) was added in three equal portions over a period of 20 min. and stirred for 3 hrs at 0°C. Then the reaction mixture was stirred at room temperature for 24 hrs (Scheme 1). The completion of reaction was eluted by Thin Layer Chromatography (TLC). After the consumption of starting material, the reaction mixture was transferred into a separating funnel and pH was adjusted to 4.5 with 1N HCl. About 25 mL of dichloromethane was added to the above reaction mixture and shaken well. Aqueous layer was separated and the organic layer was washed twice with water and finally saturated sodium chloride solution was added to the organic layer and the contents were shaken well for 10 min. Now the organic layer was filtered into a conical flask, dried using sodium sulphate and concentrated using a rotary evaporator. The crude Fmoc-α-(2,5,7-tri-tert-butylindol-3-yl)alanine was purified by column chromatography using different eluent compositions of hexane and ethyl acetate.
Peptide design and synthesis
The Fmoc-chemistry solid-phase peptide synthesis method was used in this study to synthesise the peptide using Rink amide resin. First, the rink amide resin was neutralised using a 5% DIPEA solution. To synthesise all peptides, all amino acids were linked as HBTU with 1 equivalent of HOBT and 2.5 equivalents of DIPEA. When the synthesis was finished, the Fmoc protective moiety evaporated. The resins were treated overnight with the HBTU active ester of the peptides P2 and P3-(2,5,7-tri-tert-butylindol-3-yl)alanine (W). Before drying under vacuum, the resin was carefully washed with suitable solvents such as dimethylformamide, dichloromethane, acetic acid, and diethyl ether. Trifluoroacetic acid, thioanisole, and ethanedithiol (9.0:0.5:0.5, v/v) were used to cleave the peptides from the resin for over two hours at room temperature. To obtain the crude peptide, the cleavage mixture was filtered, concentrated, and dried using diethyl ether. For all characterizations, the resulting crude peptide was centrifuged once more, dried, and kept at 4°C.
Semi-preparative HPLC purification of P2 and P3
The crude peptides were purified by using a preparative column, YMC-Pack, ODS-A, (250 × 20.0) mm, 10 µL (YMC Co., Ltd. Japan.) stainless steel column filled with octadecyl bonded porous silica gel. Waters Preparative HPLC system (Waters Corporation, USA) having gradient elution capability was used to purify the peptide. Using an analytical column, the XTerra RP-18, measuring (150×4.6) mm, 5 µL, it was possible to determine how pure the peptides were. The purity of the peptide was assessed using a Waters Alliance HPLC system (Waters Corporation, USA) with gradient elution capability. HPLC Grade milli-Q water containing 0.1% TFA was used as mobile phase-A and LCMS Grade acetonitrile containing 0.1% TFA was used as mobile phase-B. The peptide eluted was detected from the absorbance at 215 nm using Waters 2487 dual absorbance UV detector. The peptides were purified by using various gradient compositions of solvents A and B. The fractions collected were pooled together, frozen at -70°C using dilute HCl to removal residual TFA and lyophilized.
Peptide integrity by RP-HPLC and Mass spectrometric analysis
The determination of molecular mass of peptides was performed in PE Sciex, API-3000 mass spectrometer using Turbo ion source as an ionization source. The instrument was calibrated prior to measurement using poly propylene glycol (PPG). Agilent 1100 series HPLC pump and auto injector was used as a sample introduction system to mass spectrometer. About 10 µL of the peptide solutions (0.20 mg/mL) in water was injected into the mobile phase composed of 10 mM ammonium acetate and acetonitrile (50:50) mixture and an isocratic flow of 0.3 mL/min was maintained for 3 min. The peptides were analyzed from 300 amu to 2000 amu (step size set at 0.4 amu and dwell time set at 0.7 ms). The purified peptides' mass was measured using an ESI-Quad Mass spectrometer. The determination of molecular mass is crucial in determining the structural integrity of the peptides.
Circular dichrosim Spectroscopy
A Jasco (J-715) Spectropolarimeter that was calibrated with 10-(+) camphor sulfonic acid was used to record circular dichroism (CD) spectra. Each peptide was independently synthesised utilising solvents such as 20% TFE, 10 mM Sodium phosphate buffer (pH 7.4), and 30 mM SDS solution, yielding about 20 M of each in various solutions. Using a 1-mm cell, measurements were taken at 1-nm intervals in the range of 270 to 190 nm. Each sample and its equivalent control sample without peptide received an average of four scans. Contributions from the control samples were subtracted from the corresponding peptide samples and then the spectra were normalized for concentration and path length. The spectra are presented as a function of mean residue ellipticity vs. wavelength.
Preparation of small unilamellar vesicles (SUVs)
The lipid vesicles were prepared by extrusion method. A vertex tube was filled with 3 mg of lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG)) in chloroform, which was then used to remove the solvent using a stream of nitrogen gas. PBS buffer was used to rehydrate the lipids to a concentration of 3 mg/mL, and a vortex mixer was used to thoroughly mix the mixture. The multilamellar vesicles thus obtained were then extruded 8 times through a polycarbonate filter mounted inside a Acrodisc™ syringe to get a clear suspension (SUVs).
Lipid membrane binding assay
A dilute solution (5 µM, 3 mL) of each peptide (P2 and P3) in PBS solution was used for fluorescence measurements. Spectra were recorded 5 min. after adding increasing amounts of lipid vesicles. The intrinsic fluorescence of tryptophan residue was exploited for this purpose on a Fluoromax spectrofluorometer. Spectral measurements were performed by setting the excitation wavelength at 284 nm, with excitation and emission slits set to 5 nm. The steady state fluorescence emission spectra were averaged from 2 scans recorded in the range from 320 nm to 420 nm.
Antibacterial Activity Assay 20
The antimicrobial activity of P2 and P3 were determined in two steps i.e., qualitative test (disk diffusion method), and quantitative estimation (Minimum inhibitory concentration (MIC)). For disk diffusion method, sub-culture of appropriate bacterium was prepared in 100 mL LB medium and incubated at 37°C with shaking at 160 rpm for 48 h. Approximately 30 mL of the agar medium containing the appropriate bacterial strains was aseptically placed into each sterilised Petri plate. A disk of 6 mm was placed on agar surface and after that 25 µL of peptides P2 and P3 was added and with control peptide P1. The diameter of the zone of inhibition was evaluated after 24–48 hours of incubation at 37°C in antibacterial assay plates.
For MIC calculation, 96 well plates were used. In one sterile well 75 µL of peptides (control P1 and synthesized peptide P2 and P3) at concentrations ranging from 5.0 to 80 µg/mL was diluted using 75 µL of test microorganisms and 100 µL of broth medium, and then incubated at 37°C for 24–48 h. The control was made in two sets, one with broth medium and test organism and the other with broth medium and extract. After 48 h, the MIC values were determined by increase in OD600, which was measured using a BioTek EL 96-plate reader (BioTek Instruments, Winooski VT). The lowest concentration that resulted in a complete inhibition of growth was interpreted as MIC. The test was further confirmed by plating on LB agar.
Hemolysis Assay 21, 22
The buffy coat was removed after freshly obtained venous human blood in heparin-buffer was centrifuged at 2500 rpm for 5 minutes at 11°C. The acquired erythrocytes underwent three rounds of washing in PBS (10 mM phosphate, 150 mM NaCl, pH 7.4). Red Blood Cells (RBCs) (2×107) were introduced into 1.0 mL volumes of 20, 40, 60, 80, and 100 M solutions after the peptides had been diluted in PBS. Centrifugation at 2500 rpm was used to extract the unlysed RBCs after the cells had been mixed and incubated at 37°C for 30 min. By measuring the absorbance at 540 nm (Soret band), the amount of liberated hemoglobin that was present in the supernatant was tracked spectrophotometrically. In 1 mL of water, 2×107 RBCs were lysed and the absorbance of the solution at 540 nm was used for the calculation of percentage hemolysis.
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
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
Taking lead from the activities of P1, the 8-residue peptide (P2 and P3) was synthesized using Fmoc-chemistry protocols on Rink Amide Resin. The integrity of the peptides was confirmed by mass spectral analysis. The homogeneity of the peptides was established by HPLC methods. The purity of the peptides was determined by comparing the reversed phase HPLC chromatograms of the crude and purified peptides. The hydrophobicity of the peptides was determined directly from the retention times of the peptides under identical elution conditions. The conformational preferences of the peptides were studied using circular dichrosim spectroscopy in media of different polarity to assess the conformational transitions upon interaction with membranes. In contrast to the partially folded structure of P1 non-polar environments, P2 adopts an extended structure in both polar and non-polar environments. Predictably, P3 exists in an extended conformation owing to the presence of two D-tryptophan derivatives. The binding affinity of the peptides for zwitterionic and acidic lipid membranes was determined using fluorescence based binding assays to assess the membrane selective binding ability. The intrinsic fluorescence of the peptides was exploited for this purpose. While the two peptides generally showed preferential binding to negatively charged acidic membrane, peptide P3 showed exceptional affinity for both zwitterionic POPC, and anionic POPC/POPG membranes as revealed by the binding curves. The efficacies of the peptides were tested against Gram positive and Gram-negative bacteria. As expected, the most hydrophobic peptide P3 was the most active against all the test organisms. To test the toxicity of these peptides, hemolytic assay was performed using these peptides. Interestingly, P2 showed about 50% toxicity at concentrations much higher than its MIC. The most active peptide P3 was both hemolytic and antimicrobial even at lower concentrations 10 µg/mL.
In the present investigation, we have recognized two budding antimicrobial peptides i.e., P2, and P3, both the peptides were showing potential antimicrobial activity against pathogenic bacteria i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis by having MIC range from 10–50 µg/mL. The knowledge gained in this study was further useful in designing potent antimicrobial peptides, which specifies on the way to their standing in bio-prospection with respect to the industrial application for falling the antibiotics resistance. Large-scale production and exhaustive molecular explanation of these antimicrobial peptides may lead to the discovery of the novel and potential antimicrobial peptides.
ACKNOWLEDGMENTS
The authors hearty thank to Dr. S. Thennarasu, Chief Scientist, CSIR-CLRI, Adyar, Chennai, Tamilnadu, India for providing research support. There is no external funding research support for carrying out this work.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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No competing interests reported.