Short Antimicrobial Peptides: Therapeutic Potential and Recent Advancements.

There has been a lot of interest in antimicrobial peptides (AMPs) as potential next-generation antibiotics. They are components of the innate immune system. AMPs have broad-spectrum action and are less prone to resistance development. They show potential applications in various fields, including medicine, agriculture, and the food industry. However, despite the good activity and safety profiles, AMPs have had difficulty finding success in the clinic due to their various limitations, such as production cost, proteolytic susceptibility, and oral bioavailability. To overcome these flaws, a number of solutions have been devised, one of which is developing short antimicrobial peptides. Short antimicrobial peptides do have an advantage over longer peptides as they are more stable and do not collapse during absorption. They have generated a lot of interest because of their evolutionary success and advantageous properties, such as low molecular weight, selective targets, cell or organelles with minimal toxicity, and enormous therapeutic potential. This article provides an overview of the development of short antimicrobial peptides with an emphasis on those with ≤ 30 amino acid residues as a potential therapeutic agent to fight drug-resistant microorganisms. It also emphasizes their applications in many fields and discusses their current state in clinical trials.

Synergistic effects of short peptides and antibiotics against bacterial and fungal strains.

There is a rising tide of concern about the antibiotic resistance issue. To reduce the possibility of antibiotic-resistant infections, a new generation of antimicrobials must be developed. Antimicrobial peptides are potential alternatives to antibiotics that can be used alone or together with conventional antibiotics to combat antimicrobial resistance. In this work, lead compounds LP-23, DP-23, SA4, and SPO from previously published studies were synthesized by solid-phase peptide synthesis and their antimicrobial evaluation was carried out against various bacterial and fungal strains. Peptide combinations with antibiotics were evaluated by using the checkerboard method and their minimal inhibitory concentration (MIC) in combination was calculated by using the fractional inhibitory concentration (FIC) index. Cytotoxicity evaluations of these peptides further confirmed their selectivity toward microbial cells. Based on the FIC values, LP-23, DP-23, and SPO demonstrated synergy in combination with gentamicin against a gentamicin-resistant clinical isolate of Escherichia coli. For Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium, seven combinations exhibited synergistic effects between peptide/peptoids and the tested antibiotics. Additionally, almost all the combinations of peptides/peptoids with amphotericin B and fluconazole also showed effective synergy against Aspergillus niger and Aspergillus flavus. The synergy found between LP-23, DP-23, SA4, and SPO with the selected antibiotics may have the potential to be used as a combination therapy against various microbial infections.

A standardized polyherbal preparation POL-6 diminishes alcohol withdrawal anxiety by regulating Gabra1, Gabra2, Gabra3, Gabra4, Gabra5 gene expression of GABAA receptor signaling pathway in rats.

BACKGROUND: Alcohol abuse is a major problem worldwide and it affects people's health and economy. There is a relapse in alcohol intake due to alcohol withdrawal. Alcohol withdrawal anxiety-like behavior is a symptom that appears 6-24 h after the last alcohol ingestion. METHODS: The present study was designed to explore the protective effect of a standardized polyherbal preparation POL-6 in ethanol withdrawal anxiety in Wistar rats. POL-6 was prepared by mixing the dried extracts of six plants Bacopa monnieri, Hypericum perforatum, Centella asiatica, Withania somnifera, Camellia sinesis, and Ocimum sanctum in the proportion 2:1:2:2:1:2 respectively. POL-6 was subjected to phytochemical profiling through LC-MS, HPLC, and HPTLC. The effect of POL-6 on alcohol withdrawal anxiety was tested using a two-bottle choice drinking paradigm model giving animals' free choice between alcohol and water for 15 days. Alcohol was withdrawn on the 16th day and POL-6 (20, 50, and 100 mg/kg, oral), diazepam (2 mg/kg) treatment was given on the withdrawal days. Behavioral parameters were tested using EPM and LDT. On the 18th day blood was collected from the retro-orbital sinus of the rats and alcohol markers ALT, AST, ALP, and GGT were studied. At end of the study, animals were sacrificed and the brain was isolated for exploring the influences of POL-6 on the mRNA expression of GABAA receptor subunits in the amygdala and hippocampus. RESULTS: Phytochemical profiling showed that POL-6 contains major phytoconstituents like withaferin A, quercetin, catechin, rutin, caeffic acid, and ß-sitosterol. In-vivo studies showed that POL-6 possesses an antianxiety effect in alcohol withdrawal. Gene expression studies on the isolated brain tissues showed that POL-6 normalizes the GABAergic transmission in the amygdala and hippocampus of the rats. CONCLUSION: The study concludes that POL-6 may have therapeutic potential for treating ethanol-type dependence.

Recent Updates on Antifungal Peptides.

The current trend of increment in the frequency of antifungal resistance has brought research into an era where new antifungal compounds with novel mechanisms of action are required. Natural antimicrobial peptides, which are ubiquitous components of innate immunity, represent their candidature for novel antifungal peptides. Various antifungal peptides have been isolated from different species ranging from small marine organisms to insects and from various other living species. Based on these peptides, various mimetics of antifungal peptides have also been synthesized using non-natural amino acids. Utilization of these antifungal peptides is somehow limited due to their toxic and unstable nature. This review discusses recent updates and future directions of antifungal peptides, for taking them to the shelf from the bench.

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation.

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.

In vivo infection and In vitro stress survival studies of acid susceptible mutant of Mycobacterium fortuitum.

Background: Ubiquitous presence of Mycobacterium fortuitum and ability to cause infections in human beings, hints toward its integral resistance against environmental and host stress conditions. With an aim to identify genes responsible for adapting in vitro acidic stress of M. fortuitum, in the previous study, TnphoA random mutagenesis identified acid susceptible mutant MT727, with mutation in ribosomal maturation factor encoding gene rimP, to be mutated. The present study was conducted to explore virulent behavior as well as growth behavior under in vitro stress conditions. Methods: Acid susceptible transposon mutant MT727 was injected intravenously in female BALB/c mice and kidney tissue was analyzed for the bacillary load as well as pathological characterization. Cytokine profiling of MT727-infected mice serum was done. MT727 was also subjected to various in vitro stress conditions, including detergent stress, heat stress, and hypoxic stress. The viable count of bacteria under different stress conditions was determined at regular time interval. Results: Mutant MT727 showed slight variation in bacillary load in vivo; however, defective growth behavior under detergent and hypoxic stress was observed when compared to wild type strain. Conclusion: Results conclude probable involvement of rimP gene in survival of M. fortuitum under hypoxic stress and detergent stress conditions.

Cationic antimicrobial peptide and its poly-N-substituted glycine congener: Antibacterial and antibiofilm potential against A. baumannii.

Acinetobacter baumannii is one of the clinically important nosocomial pathogen that has become resistant to most of the conventional antimicrobials. Biofilms formed by A. baumannii are difficult to eradicate, thereby highlighting the need for new therapeutic options to treat biofilm associated infections. Antimicrobial peptides have recently emerged as new alternatives to conventional antibiotics, but peptides often suffer with drawbacks such as poor proteolytic stability and high cost of production. To tackle these limitations, mimetics based on antimicrobial peptides are usually designed and synthesized. In this study we have designed and synthesized a peptoid based on a minimum amphipathic template of a twelve residue cationic peptide. Antimicrobial evaluation of peptide and peptoid was carried out against biofilm producing A. baumannii strains. Further, proteolytic stability study of these compounds was carried out in human serum and morphological alterations caused by them on A. baumannii were visualized by SEM analysis. In addition, these compounds were found to be non toxic to human erythrocytes at their minimum inhibitory concentrations against A. baumannii strains. Overall results obtained in this study suggest that these compounds might be potential antimicrobial agents against biofilm forming A. baumannii and it may be postulated that their mode of action on A. baumannii is disruption of bacterial cell membrane.

Ribosomal maturation factor (RimP) is essential for survival of nontuberculous mycobacteria Mycobacterium fortuitum under in vitro acidic stress conditions.

Mycobacterium fortuitum is an important human pathogenic NTM, which resists stress conditions inside macrophages by exploitation of specific genes. TnphoA-based transposon mutagenesis was employed to identify membrane genes responsible for survival of M. fortuitum under such stress conditions. A library of about 450 mutants was constructed after electroporation of vector pRT291 into wild-type M. fortuitum. On the basis of blue color development and alkaline phosphatase assay, 20 mutants were shortlisted to screen for growth and survival under acidic stress at pH 6.5, 5.5, 4.5, and 3.5. Mutant MT727 showed reduced growth and survival under acidic stress. The acid susceptible mutant MT727 was subjected to other in vitro stress conditions prevalent inside macrophages including oxidative, nutrient starvation and nitrosative stress. However, the mutant showed no appreciable difference in growth behavior under oxidative, nutrient starvation and nitrosative stress conditions in comparison to the wild type. Genomic and bioinformatics analysis of MT727 led to identification of putative ribosomal maturation factor RimP of M. fortuitum to be affected by mutagenesis, showing closest homology to M. abscessus RimP. In silico functional interaction of RimP protein using STRING database showed its interaction with proteins of ribosomal assembly and maturation. Results indicate role of rimP gene in survival of M. fortuitum under acidic stress conditions which may be further explored for use as a potential drug target against M. fortuitum and other mycobacterial infections.

Design and synthesis of cell selective α/ß-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus.

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or ß-amino acids. Sequence A-12 was protease susceptible whereas its α/ß-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2-32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/ß-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents.

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug-resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK-52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5-4.5 µg/mL. Calcein dye leakage experiments with model membrane suggested the membrane-active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next-generation antimicrobial therapy.

Development of novel membrane active lipidated peptidomimetics active against drug resistant clinical isolates.

A new series of small cationic lipidated peptidomimetics have been synthesized and found to be highly active against several susceptible as well as drug resistant clinical isolates of bacteria and fungi. All lipidated peptidomimetics do not cause significant lysis of human erythrocytes (HC50>200µg/mL). Calcein dye leakage experiment revealed membranolytic effect of LPEP08 which was further confirmed by scanning electron microscopy (SEM). The involvement of intracellular targets as an alternate mode of action was precluded by DNA retardation assay. Additionally, LPEP08 exhibit high proteolytic stability and dose not elicit resistance against drug resistant clinical isolate of Staphylococcusaureus, even after 16 rounds of passaging. These results demonstrate the potential of lipidated peptidomimetics as biocompatible anti-infective therapeutics.

Antibacterial evaluation of structurally amphipathic, membrane active small cationic peptidomimetics: synthesized by incorporating 3-amino benzoic acid as peptidomimetic element.

A new series of small cationic peptidomimetics were synthesized by incorporating 3-amino benzoic acid (3-ABA) in a small structural framework with the objective to mimic essential properties of natural antimicrobial peptides (AMPs). The new design approach resulted into improvement of activity and selectivity in comparison to linear peptides and allowed us to better understand the influence of structural amphipathicity on biological activity. Lead peptidomimetics displayed antibacterial activities against resistant pathogens (MRSA & MRSE). A calcein dye leakage experiment revealed a membranolytic effect of 4g and 4l which was further confirmed by fluorescence microscopy. In addition, proteolytic stability and no sign of resistance development against Staphylococcus aureus and MRSA demonstrate their potential for further development as novel antimicrobial therapeutics.

In vitro and in vivo antibacterial evaluation and mechanistic study of ornithine based small cationic lipopeptides against antibiotic resistant clinical isolates.

We investigated the in vitro activities of short lipopeptides against a large panel of clinical isolates of antibiotic resistant bacteria. In the animal model, LP16 (5 mg/kg) significantly decreased the burden of viable colony forming unit (CFU) of bacteria. MTT assay results revealed the high selectivity of lipopeptides toward microbial cells. Calcein dye leakage experiments and flow cytometric analysis suggests the membranolytic effect of lipopeptides, which was further confirmed by visualizing bacterial damage via electron microscopy tool (SEM & TEM). Moreover, stability in human blood plasma and no sign of resistance development against clinical isolates of Escherichia coli and Staphylococcus aureus were observed for lead lipopeptides. These results demonstrate the potential of short lipopeptides as a novel class of anti-infectives.

Systematic study of non-natural short cationic lipopeptides as novel broad-spectrum antimicrobial agents.

We describe the design and synthesis of a new series of non-natural short cationic lipopeptides (MW = 700) as antimicrobial agents. All of the synthesized lipopeptides were tested against a range of microbes such as Gram-positive, Gram-negative bacteria, fungi including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). By systematic study of design template, we found that three ornithine residues conjugated with myristic acid are minimum requirement for a compound to be an antimicrobial agent. The most potent lipopeptide LP16 possesses broad-spectrum antimicrobial activity and has MICs in the range of 1.5-6.25 µg/mL against Escherichia coli, S. aureus, Pseudomonas aeruginosa, Bacillus subtilis, and MRSE. All lipopeptides showed high selectivity toward microbial strains as compared to human red blood cells (HC50 > 250 µg/mL). Moreover, most potent lipopeptides (LP16 and LP23) did not induce drug resistance in S. aureus even after 15 rounds of passaging. In addition, a representative lipopeptide (LP16) showed tryptic stability for 24 h. These results suggest the potential of short cationic lipopeptides to boost the discovery of future antimicrobial therapeutics.

Small cationic antimicrobial peptidomimetics: emerging candidate for the development of potential anti-infective agents.

Rapid increase in the emergence and spread of microbes resistant to conventionally used antibiotics has become a major threat to global health care. Antimicrobial peptides (AMPs) are considered as a potential source of novel antibiotics because of their numerous advantages such as broad-spectrum activity, lower tendency to induce resistance, immunomodulatory response and unique mode of action. However, AMPs have several drawbacks such as; susceptibility to protease degradation, toxicity and high costs of manufacturing. Therefore, extensive research efforts are underway to explore the therapeutic potential of these fascinating natural compounds. This review highlights the potential of small cationic antimicrobial peptidomimetics (SCAMPs; M.W. ≅ 700 Da) as new generation antibiotics. In particular, we focused on recently identified small active pharmacophore from bulky templates of native AMPs, ß-peptides, and lipopeptides. In addition, various design strategies recently undertaken to improve the physicochemical properties (proteolytic stability & plasma protein binding) of small cationic peptides have also been discussed.

Recent Approaches in design of Peptidomimetics for Antimicrobial Drug Discovery Resear.

Resistant pathogenic microbial strains are emerging at a rate that far exceeds the pace of new anti-infective drug development. In order to combat resistance development, there is pressing need to develop novel class of antibiotics having different mechanism of action in comparison to existing antibiotics. Antimicrobial peptides (AMPs) have been identified as ubiquitous components of innate immune system and widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from broad spectrum of activity to low propensity of resistance development. However, AMPs present several drawbacks that strongly limit their clinical applicability as ideal drug candidates such as; poor bioavailability, potential immunogenicity and high production cost. Thus, to overcome the limitations of native peptides, peptidomimetic becomes an important and promising approach. The different research groups worldwide engaged in antimicrobial drug discovery over the past decade have paid tremendous effort to design peptidomimetics. This review will focus on recent approaches in design of antimicrobial peptidomimetics their structure-activity relationship studies, mode of action, selectivity & toxicity.

Recent approaches in design of peptidomimetics for antimicrobial drug discovery research.

Resistant pathogenic microbial strains are emerging at a rate that far exceeds the pace of new anti-infective drug development. In order to combat resistance development, there is pressing need to develop novel class of antibiotics having different mechanism of action in comparison to existing antibiotics. Antimicrobial peptides (AMPs) have been identified as ubiquitous components of innate immune system and widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from broad spectrum of activity to low propensity of resistance development. However, AMPs present several drawbacks that strongly limit their clinical applicability as ideal drug candidates such as; poor bioavailability, potential immunogenicity and high production cost. Thus, to overcome the limitations of native peptides, peptidomimetic becomes an important and promising approach. The different research groups worldwide engaged in antimicrobial drug discovery over the past decade have paid tremendous effort to design peptidomimetics. This review will focus on recent approaches in design of antimicrobial peptidomimetics their structure-activity relationship studies, mode of action, selectivity & toxicity.

Comparative mode of action of novel hybrid peptide CS-1a and its rearranged amphipathic analogue CS-2a.

Cell selective, naturally occurring, host defence cationic peptides present a good template for the design of novel peptides with the aim of achieving a short length with improved antimicrobial potency and selectivity. A novel, short peptide CS-1a (14 residues) was derived using a sequence hybridization approach on sarcotoxin I (39 residues) and cecropin B (35 residues). The sequence of CS-1a was rearranged to enhance amphipathicity with the help of a Schiffer-Edmundson diagram to obtain CS-2a. Both peptides showed good antibacterial activity in the concentration range 4-16 µg·mL(-1) against susceptible as well as drug-resistant bacterial strains, including the clinically relevant pathogens Acenatobacter sp. and methicillin-resistant Staphylococcus aureus. The major thrust of these peptides is their nonhaemolytic activity against human red blood cells up to a high concentration of 512 µg·mL(-1). Compared to CS-1a, amphipathic peptide CS-2a showed a more pronounced α-helical conformation, along with a better membrane insertion depth in bacterial mimic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) small unilamellar vesicles. With equivalent lipid-binding affinity, the two peptides assumed different pathways of membrane disruption, as demonstrated by calcein leakage and the results of transmission electron microscopy on model bacterial mimic large unilamellar vesicles. Extending the work from model membranes to intact Escherichia coli cells, differences in membrane perturbation were visible in microscopic images of peptide-treated E. coli. The present study describes two novel short peptides with potent activity, cell selectivity and divergent modes of action that will aid in the future design of peptides with better therapeutic potential.

Usefulness of organic acid produced by Exiguobacterium sp. 12/1 on neutralization of alkaline wastewater.

The aim of this study was to investigate the role of organic acids produced by Exiguobacterium sp. strain 12/1 (DSM 21148) in neutralization of alkaline wastewater emanated from beverage industry. This bacterium is known to be able to grow in medium of pH as high as pH 12.0 and to neutralize alkaline industrial wastewater from pH 12.0 to pH 7.5. The initial investigation on the type of functional groups present in medium, carried out using FT-IR spectroscopy, revealed the presence of peaks corresponding to carbonyl group and hydroxyl group, suggesting the release of carboxylic acid or related metabolic product(s). The identification of specific carboxylic group, carried out using RP-HPLC, revealed the presence of a single peak in the culture supernatant with retention time most similar to formic acid. The concentration of acid produced on different carbon sources was studied as a function of time. Although acid was present in same final concentration, the rate of acid production was highest in case of medium supplemented with sucrose followed by fructose and glucose. The knowledge of metabolic products of the bacterium can be considered as a first step towards realization of its potential for large-scale bioremediation of alkaline wastewater from beverage industry.

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775.

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 mug/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.