Antibacterial activity of the designed peptide systems (MS1 to MS6), against Staphylococcus aureus, Escherichia coli and Gentamicin-resistant MRSA. Comparative activity assessment of antimicrobial activity of the designed peptides calculated as Minimal Inhibitory Concentration (MIC) of peptides against both M.
Acknowledgements
ABSTRACT
Tuberculosis is one of the leading causes of death, with an annual death rate of 2 million. Stability against enzymatic degradation was one of the main reasons for not using peptides, which could otherwise be a good therapeutic option with minimal side effects.
Introduction
Gramicidin, oxytocin, vasopressin and angiotensin were few molecules used in the early years of peptide-based therapeutics 1. However, the progression of peptides as a clinical agent has been set back due to the repeated failures in the later phases of clinical trials .
Stereo-chemical Engineering of Peptides with Diversified Tacticity and their Potential as future
Antibiotics
Antimicrobial peptides
A source of antimicrobial peptides found at different levels of organisms, according to their relative occurrence. The presence of a net negative charge in the bacterial membrane is one of the fundamental causes of the antimicrobial properties of cationic antimicrobial peptides 61 .
Characteristics of antimicrobial peptides
Some reported examples where bacterial and other expression systems have shown high-volume production of antimicrobial peptides can be found in related references 132-134. The reduced bioavailability of peptide molecules can be attributed to their respective pharmacokinetic properties [absorption, distribution, metabolism and excretion (ADME)] 5 (Figure 2.5).
Antibiotic peptides with diversified tacticity
- Mechanism of action
- Modeling Peptide – Membrane Interaction
The interaction of peptide molecules varies largely depending on the structure and chemistry of the membrane. However, pore stability is concentration dependent, indicating the inability of peptide activity below a threshold value of 170, 171.
Research Design
Amino acids can be divided into polar, non-polar, aliphatic, aromatic, negatively or positively charged, etc. The main objective of this thesis work is the design synthesis and characterization of a new genre of antimicrobial peptides incorporating amino acids with L and D stereochemistry.
Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides
Introduction
In this work, we present a design philosophy to synthesize cationic, amphipathic peptide molecules incorporating D-amino acids in the design alphabet. The bactericidal potency of the designed peptides was higher against Gram-positive than Gram-negative bacteria, although the design approach may be further modified in future designs in the generation of broad-spectrum antibiotics.
Materials and methods .1. Reagents and chemicals
- Methodology
- Modelling and simulation
- Electrostatic profiling
- Peptide synthesis and characterization
- Anti-bacterial assay
- Field Emission-Scanning Electron Microscopy (FE-SEM)
- Hemolytic activity
The resulting suspension was diluted to a net absorbance value of 0.2 at 600 nm and 50 µL of the inoculum was treated with the required peptide concentrations (net incubation broth is 100 µL), followed by 2 hours of incubation. 50 µL of cell suspensions were mixed with the required concentration of peptides and incubated for 2 h at 37±2 °C.
Results and Discussion
- Antibacterial assay and toxicity studies: The in vitro antimicrobial activity of the synthesized peptides was tested against Gram-positive (Staphylococcus
- Hemolytic activity: Hemolytic assay against mammalian RBC was performed to estimate the extent of toxicity. All four peptides exhibited negligible cytotoxicity
- Microscopic analysis: A qualitative evaluation of membrane rupturing potential of designed antimicrobial peptide against bacteria was performed using
Electrostatic potential maps of the designed peptides were generated by solving FDPBE using Delphi software. Minimum inhibitory concentration of the designed peptides against S.aureus and P.aeruginosa shows preferential activity of syndiotactic sequences against Gram-positive bacteria. To verify the stability of the designed peptides in a 6,3-helix conformation, similar to gramicidin helix, a 10 ns MD was performed at 298 K under NVT conditions using GROMOS 96.
Electrostatic potential map of designed peptides using Delphi software solving the finite-difference Poisson-Boltzmann equation. In a previous study 7 we systematically varied the stereochemistry of the designed peptides, resulting in different structures and electrostatic interaction profiles while approaching the target.
Effect of tacticity-derived topological constraints in bactericidal peptides
Materials and methods
- Peptide-lipid interaction
The magnitude of the interaction is measured in terms of the change in surface pressure (mN m-1) of the system. Estimation of Saturated Peptide Concentration: Surface pressure was monitored by the Wilhelmy method, using a paper plate and a custom-made polytetrafluoroethylene (PTFE) Langmuir trough (approximately 15 cm3 volume) run on a Langmuir instrument (Biolin Scientific, UK). Peptides were injected into 10 mL subphase (10 mM phosphate buffer, pH 7.4) and surface pressure changes were monitored until saturation was reached.
After evaporation of the chloroform and stabilization of the pressure, peptides at a saturated concentration were steadily injected into the subphase (10 mM phosphate buffer, pH 7.4) through the vertical hole to cause minimal disruption of the lipid layer. Interactions with peptide monolayers were recorded as changes in the surface pressure of the monolayers and the resulting data were plotted as change in surface pressure (mN m-1) versus time (minutes).
Results and Discussion
- Modeling, simulation and electrostatic profiling
- Peptide-lipid interaction: Differential topology dependent electrostatic profiles of the sequences give insight to test whether they differ in their
- Field Emission Scanning Electron Microscopy (FE-SEM): To provide a topographical view of the bacterial membrane perturbation by the designed
The electrostatic potential was calculated by solving the Poisson-Boltzmann finite difference equation using Delphi software, summed for each amino acid side chain and displayed collectively at the chromophore center of the side chain. The interaction of the peptides was studied with monolayers consisting of POPC (zwitterionic lipid) and 7:3 POPC:POPG (negatively charged). Injection of the peptides into the subphase caused an increase in surface pressure.
Fractured (treated) and intact (untreated) membrane bacterial samples were observed, qualitatively confirming the membranolytic activity of the designed peptides (Figure 5.8). Membrane deformation due to bactericidal activity of the designed peptides against (A) Staphylococcus aureus, (B) Escherichia coli and (C) Gentamicin-resistant MRSA at their respective minimum inhibitory concentrations.
Conclusion
Bacterial cell systems consist of a negatively charged plasma membrane, with the complexity of the membrane varying from Gram-positive to Gram-negative bacteria. The Gram-positive cell wall consists of a 20-80 nm thick layer of peptidoglycans and is rich in lipoteichoic acid and its derivatives, with teichoic acid imparting most of the negative charge to the cell surface. 69 In contrast to lipoteichoic acid from Gram-positive bacteria, the negative charge on the Gram-negative bacterial surface is due to the presence of lipopolysaccharide.
The designed peptides show better activity against antibiotic-resistant Gram-positive species compared to the native Gram-positive one. This can again be attributed to the retention of the designed amphiphilicity of the syndiotactic variants and the relatively fluid nature of the homochiral sequences.
Bactericidal potency and extended serum life of stereo- chemically engineered peptides against Mycobacterium
- Introduction
- Materials and methods
- Antimicrobial assay (Micro-dilution method)
- Results and Discussion
- Design and Synthesis of Syndiotactic Antimicrobial Peptides
- Effect of stereo-chemical engineering of the designed peptides in extending plasma half life
- Conclusion
Then, 50 µL of the diluted inoculum was mixed with fixed concentrations of peptides and incubated at 37 °C for 72 hours. The syndiotactic arrangement of the peptide sequences results in the helical structure of gramicidin with alternating L and D residues in the peptide sequence. Evaluation of the comparative antimicrobial activity of the designed peptides, calculated as the minimum inhibitory concentration (MIC) of the peptides against M.smegmatis and E.coli in human serum and media alone.
The bactericidal activity of the peptide molecules was determined using the Brothmicro dilution method (Figure 6.3). To verify the potential toxicity of the designed peptides to mammalian cells, a hemolytic assay was performed with RBCs, incubating with 8 µM and 16 µM peptides for 2 h.
Effect of chain length in Modulating bactericidal potential in short syndiotactic peptides
Materials and methods
Modeling and simulation, Electrostatic profiling, Peptide synthesis and characterization, Field Emission Scanning Electron Microscopy (FE-SEM) and Haemolytic activity were performed as explained in Chapter 4 section 4.2.
Results and Discussion
- Electrostatic profiling and MD simulation of peptide sequences
- Antimicrobial assay and Hemolytic activity
- Microscopic analysis (FE-SEM)
The amphipathic nature of the de-novo designed peptides was confirmed by electrostatically mapping the potential of the designed peptides (Figure 7.2). The N- and C-terminus of the peptides are always shown together with their peptide sequences. To check the stability of the peptide candidates, a 10 ns MD simulation run was performed under NVT conditions using the GROMOS 96 force field.
The in vitro antibacterial potency of the synthesized peptides was tested against Staphylococcus aureus (Gram positive), Escherichia coli (Gram negative) and. To assess the toxicity of the designed AMPs against mammalian cells, we performed a hemolytic activity assay of peptides against mammalian red blood cells (RBC).
Conclusion
Chain length and bactericidal potential bacterial cells (S.aureus, E.coli and Gentamicin-resistant MRSA) were treated with their respective MIC values followed by their fixation and imaging. The comparative qualitative analysis of peptide-treated bacterial cells showing deformed membrane architecture compared to the untreated bacterial cells, with non-deformed membrane morphology, supports their likely membranolytic activity, although to a lesser extent compared to the 12 mer sequences. Field Emission Scanning Electron Microscopy (FE-SEM) of bacterial cells treated with peptides (ST1 – ST4) at 100 µM concentration and untreated cells as control.
Bacterial cells of Staphylococcus aureus, Escherichia coli, and gentamicin-resistant MRSA were used in this study, and all peptide-treated cells were observed to develop a deformed membrane texture compared to untreated control cells. The experiments described in this chapter suggest two main design guidelines; i) the bactericidal activity of the syndiotactic sequence depends on the length and structure of the chain and ii) the bactericidal activity is a function of the electrostatic environment created by the stereochemical sequence of amino acids and their resulting topology.
Optimization of bactericidal potential with designed electrostatic gradients
Materials and methods
Modeling, peptide synthesis, characterization, field emission scanning electron microscopy (FE-SEM) and hemolytic activities were performed as explained in chapter 4 section 4.2. Antimicrobial assay and sensitive serum assay was performed as described in Chapter 6, section 6.2.1 and 6.2.2.
Results and Discussion
- Design and Synthesis of Antimicrobial Peptides
- Antibacterial activity and serum sensitivity of designed Antimicrobial Peptides
Peptides AS01 – AS04, AS06 and AS07 had longitudinal divisions of hydrophobic (gray) and cationic (blue) zones, whereas AS05 and AS08 were designed to have hydrophobic and cationic zones in vertical divisions (Figures 8.1 and 8.2). The purified peptides were characterized by Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI-TOF) mass spectrometry. The antibacterial activity of peptides was performed against Escherichia coli, Methicillin-resistant MRSA and Mycobacterium smegmatis.
Peptide code and their respective MIC values against E.coli, Gentamicin-resistant MRSA and M.smegmatis in presence and absence of serum. All the designed peptides were good in terms of their antimicrobial activity except AS05 and AS06, and can be considered as potential candidates for further development and clinical trials.
Conclusion and Future Work
Defensins Knowledge Base: A manually curated database and information resource focused on the defensins family of antimicrobial peptides. In vitro and in vivo activities of antimicrobial peptides developed using an amino acid-based activity prediction method.
Patent and Publications
Bactericidal potency and extended serum life of stereochemically engineered peptides against Mycobacterium. International Journal of Peptide Research and Therapeutics.
Bactericidal Potency and Extended Serum Life of Stereo-Chemically Engineered Peptides Against Mycobacterium
Comparative evaluation of the antimicrobial activity of the designed peptides calculated as the Minimal Inhibitory Concentration (MIC) of the peptides against both M. This stereo-specificity shows the possibility of diversifying the basic alphabet of the amino acid sequence including D-enantiomers[13 ]. Injection of peptides into the subphase caused an increase in surface pressure.
For comparison, ruptured (treated) and intact (untreated) membrane bacterial samples were observed, qualitatively confirming the membraneolytic activity of the designed peptides (Fig. 4). 3. Antibacterial activity of the designed peptide systems (MS1 to MS6), against Staphylococcus aureus, Escherichia coli and Gentamicin-resistant MRSA.
Prakash Kishore Hazam, Gaurav Jerath, Nitin Chaudhary & Vibin Ramakrishnan
If you wish to archive your article yourself, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version into a repository, provided that it is not made public until 12 months after official publication or later and provided that the original publication source is cited and a link is included to the published article on the website of Springer.
Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides
Electrostatic potential maps of the designed peptides were generated by solving the Finite Difference Poisson Boltzmann equation using Delphi software. 2 Electrostatic potential mapping of engineered peptides, using Delphi Software solving the Poisson Boltzmann finite difference equation. In a previous study (Hazam et al. 2017), we have systematically varied the stereo-chemistry of designed peptides resulting in different structures and electrostatic interaction profiles as they approach a target.
Six model systems represent all design variables in the best possible way. The chirality program gives the side chain orientation of an amino acid residue as Dextro or Levo-rotatory.
![Figure 2.8. A schematic representation of commonly cited mechanism of action of anti-microbial peptides.[A)Barrel stave B)Carpet C)Toroidal pore andD)Disordered toroidal]](https://thumb-ap.123doks.com/thumbv2/azpdfnet/10545226.0/38.892.177.723.319.858/schematic-representation-commonly-mechanism-microbial-peptides-toroidal-disordered.webp)
