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International Journal of Peptide Research and Therapeutics

https://doi.org/10.1007/s10989-017-9650-0

and Therapeutics https://doi.org/10.1007/s10989-017-9650-0 Peptide Therapeutics Versus Superbugs: Highlight on Current

Peptide Therapeutics Versus Superbugs: Highlight on Current Research and Advancements

Krishnanand Nagarajan 1

Current Research and Advancements Krishnanand Nagarajan 1 · Sathish Kumar Marimuthu 1 · Selvamani Palanisamy 1

· Sathish Kumar Marimuthu 1 · Selvamani Palanisamy 1 · Latha Subbiah 1

Accepted: 13 November 2017 © Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract Antibiotics have saved several millions of lives, but its persistent use of antibiotics in the treatment of various infections, whether bacterial, fungal, viral or parasitic has lead to the development of antibiotic resistance. The rapid emergence of antibiotic resistant strains poses a serious challenge to existing antimicrobial therapies. Due to the increase in drug-resistant pathogens and failure of antibiotics the urgent need for the discovery of novel antimicrobials has been continuously empha- sized in the global forum. Here we review about antimicrobial peptides (AMPs), their structural insights and recent develop- ments. We had summarized the major classes, mechanism of action and biophysical parameters that modulate therapeutic potency of AMPs. Also, we had briefed the challenges involved in developing therapeutic peptides and the global market potential for peptide therapeutics.

Keywords Antimicrobial peptide · Therapeutic · Resistance · Mechanism · Biophysical parameters · Global market

Introduction

Antimicrobial resistance (AMR) is one of the most serious health threats emerging globally. This poses the greatest challenge in treating common infectious diseases; endanger public health and efficacy of antibiotics, which results in patient morbidity, mortality and increased health care costs (Izadpanah and Khalili 2015; Long et al. 2017; Elliott et al. 2017). This escalating threat to global public health requires immediate attention and action across all parts of the world. An ever-increasing rate in the degree of resistance and com- plexity of infections today has questioned the credibility and effectiveness of current antibiotics in treating severe infec- tions (Ventola 2015; Harten et al. 2017). In the early of 2017, WHO has published a global prior- ity pathogen list containing 12 drug-resistant bacteria, for which new antibiotics are urgently required. This report has insisted the importance and pressing need for developing new antibiotics against multi-drug resistant gram-negative

* Krishnanand Nagarajan krishwrites@gmail.com

1 Department of Pharmaceutical Technology, University College of Engineering, Anna University, Bharathidasan Institute of Technology Campus, Tiruchirappalli, Tamil Nadu 620024, India

Technology Campus, Tiruchirappalli, Tamil Nadu 620024, India bacteria. The threat levels were categorized based on spe-

bacteria. The threat levels were categorized based on spe- cies and the type of resistance: critical, high and medium. Carbapenem-resistant Acinetobacter baumannii, Pseu- domonas aeruginosa and carbapenem cum 3rd generation cephalosporin resistant Enterobacteriaceae (Klebsiella pneumonia, Escherichia coli, Enterobacter sp., Serratia sp., Proteus spp., and Providencia sp., Morganella sp.) respectively were stacked in the critical tire. It should also be noted that in a similar kind of report published by CDC, USA in 2013, Clostridium difficile, Carbapenem-resistant Enterobacteriaceae and Neisseria gonorrhoeae were listed in the urgent category, requiring more attention, monitoring and prevention activities (Antibiotic/Antimicrobial 2017). A very recent research has discovered that Klebsiella species is a major causative in most morbidity and mortality cases in a global scale and is a potent threat to public health. It has been noticed that many strains of this bacteria are increas- ingly gaining resistance, thus making infections much harder to treat (Antibiotic/Antimicrobial 2017; Long et al. 2017). Randomized and improper administration of antibiotics is the primary cause for the development of drug-resistance pattern in bacteria. Overusing or misusing antimicrobial drugs make resistance to develop at a faster pace. Patient non-adherence to prescribed medications, non-therapeutic use of antibiotics in livestock, poultry and agricultural operations adds as an additional reason for the development

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International Journal of Peptide Research and Therapeutics

of resistant to antibiotics (Elliott et al. 2017). Thus the growing antibiotic resistance and the decline in the rates of novel drug discovery, drive the researchers to investigate on new non-chemical antibacterial drugs to combat infectious diseases, especially those caused by multidrug-resistant (MDR) bacteria’s (Hoagland et al. 2016). It is definite that Antimicrobial peptides (AMPs) will emerge as promising alternatives to conventional antibiotics, owing to their rela- tively simple methods of synthesis and modification. Recent advancements in biotechnology have enabled synthesis of precise AMPs (Elliott et al. 2017; Hoagland et al. 2016). Antimicrobial peptides (AMPs) play a vital role in the defense mechanisms of most organisms. They were also referred to as “host defense peptides” in higher eukary- otes (vertebrates, invertebrates, and plants). Antimicrobial peptides contribute to the innate immunity. These peptides were usually constituted with 5–40 amino acids and have molecular weight of less than 10 kDa (Hoagland et al. 2016; Patel and Akhtar 2017). They function as natural antibiotics and evade the infectious pathogens. Currently, many stud- ies have reported novel functions such as skin regeneration, antineoplastic effects, anti-tumor, wound healing, and others. This review will provide a brief overview on current updates regarding AMPs as therapeutics (Figs. 1, 2, 3 and 4).

Structural Insight and AMPs Classes

AMPs universally display similar fundamental properties such as cationic nature, amphipathicity, and hydrophobicity; thus it’s possible to classify them only based on their second- ary structure. Based on the types of secondary structures and Boman’s classification AMPs are grouped into four families:

α, β, αβ, and non-αβ respectively. Further, the Eukaryotic

β, αβ, and non-αβ respectively. Further, the Eukaryotic Fig. 1 Briefing, the timeline of antibiotic deployment

Fig. 1 Briefing, the timeline of antibiotic deployment and resistance, observed. Source: Centers for Disease Control and Prevention, US Department of Health and Human Services, Atlanta, GA, 2015

Department of Health and Human Services, Atlanta, GA, 2015 Fig. 2 Structural diversification of 2818 AMPs

Fig. 2 Structural diversification of 2818 AMPs in the APD database, Source: Antimicrobial Peptide Database—UNMC, (http://aps.unmc. edu/AP/main.php)

antimicrobial peptides can be categorized into cationic (e.g.:

defensins, cathelicidins, cecropins, thionins) and anionic (Maximin, dermcidin) peptides.

Type I (α‑Helical Peptides)

Alpha-helical peptides are the abundant and widespread class of cytolytic AMPs with amphipathic and alpha-helical domains that enable efficient interactions with the biologi- cal membrane. Cecropin B, Cap11, Melittin, Cecropin P1, Cap18, and magainins are some of the well-known peptides of this class (Ebbensgaard et al. 2015). The membrane-bind- ing motif of an amphipathic helix is formed by a sequence of linear amino acid chains with a recurrence of polar and apolar residues for every three to four residues along the structure. This structure, with the polar side chains aligned along one side and the hydrophobic residues along the oppo- site side of the helical coat, allows an optimal interaction of these peptides with the amphiphilic structure of the biologi- cal membrane. The majority of the AMPs characterized so far belong to this group of α-helical peptides. Hydrophobic- ity and helicity are critical parameters that govern the bio- logical activities of α-helical peptides. The hydrophobicity and helicity of a peptide can be modulated by d -amino acid substitution approach to form d - and l -diastereomeric pep- tides, which in turn exhibit an increased peptide specificity, stability, stronger antimicrobial activity and lower cytotoxic- ity against healthy cells (Huang et al. 2014).

Type II (Beta Sheet Containing Peptides)

The beta pleated peptide family usually contains small, cysteine-rich β-strands containing peptides and are widely distributed in the animal kingdom. AMPs with beta-strands are semi or cyclic molecules whose structures are stabilized by one to three intramolecular cysteine disulfide bridges. Molecules of the β-sheet peptide families include defensins, tachyplesins, protegrins, bactenecin, dodecapeptides and

International Journal of Peptide Research and Therapeutics

International Journal of Peptide Research and Therapeutics Fig. 3 Modes of action of antimicrobial peptides. AMPs

Fig. 3 Modes of action of antimicrobial peptides. AMPs exert direct neutralizing effects on bacterial pathogens which result in membrane disruption or act upon internal targets of bacterial cells. In addition to the direct mode of action, AMPs also mediate cells for acquired

direct mode of action, AMPs also mediate cells for acquired Fig. 4 Distribution of FDA approved

Fig. 4 Distribution of FDA approved therapeutic peptides and pro- teins based on routes of administration. Source: THPdb database (http://crdd.osdd.net/raghava/thpdb/roa.php)

others (Miyasaki and Lehrer 1998). These peptides range between 16 and 18 residues in length and exhibit broad spec- trum antimicrobial activity and appear to function by mem- brane disruption. They were also found to possess antiviral activity against HIV-1 (Sitaram and Nagaraj 1999). Mol- luscan defensins have initially been characterized in mussel species, and a variety of different defensins were also then

immunity (neutrophils, t-cells, macrophages) to regulate inflam- mation and increase bacterial clearance. Adapted from Jirku et al. (2015). Antimicrobial peptides in human sepsis

reported in oysters, this includes the Cysteine-stabilized αβ defensin and big defensin AMP families (Schmitt et al. 2016). Structure–function relationship studies in defensins have revealed that the number of disulfide bridges and the cysteine spacing has a significant effect on the antimicrobial efficacy (Sharma and Nagaraj 2015).

Type III (Peptides with Over‑representation of Certain Amino Acids)

A limited number of AMPs fall under this class of peptides and usually differ from other classes due to lack of classical secondary structure. These peptides distinguish itself with an unusually high content of a specific amino acid, often proline, tryptophan, histidine, and arginine. The conforma- tions of these peptides are characterized by having minimal structural constraints and adapt more extended structures. Penaeidins, crustacean antimicrobial peptide with molecular masses ranging from 5.5 to 6.6 kDa, is characterized by an over-representation of proline residues in their NH2-terminal domain and by six cysteine residues in their COOH-terminal domain (Destoumieux et al. 1997). Mytimacin-AF is another

International Journal of Peptide Research and Therapeutics

such kind of peptide characterized from a land-living mol- lusk A. fulica, a novel cysteine-rich, cationic peptide com- posed of 80 amino acid residues which include ten cysteines (Zhong et al. 2013). The unusual amino acid composition of these AMPs has directed the researchers to set down the role of multiple tryptophan residues in its biological activity as well as its interactions with model biological membranes (Pasupuleti 2009).

Type IV (Peptides with Looped Structure and Single Bond)

This class of peptides is characterized by their looped struc- ture imparted by the presence of a single bond (disulfide or amide or isopeptide bond). This class of peptides differs from the Type II peptides in having only single disulfide bond and antiparallel β-sheet orientation. Lantibiotics belonging to this class are extensively studied due to their unique biochemistry, genetic regulation, and a variety of bio- logical functions (Pasupuleti 2009). The loop AMPs (e.g., bactenecin), adopt a loop formation with one disulfide bridge (Seo et al. 2012). Thanatin, a 21-residue antimicrobial pep- tide, similar to brevinin family contain C-terminal disulfide loop exhibit broad range of activity against bacteria. This class of peptides holds considerable potential to combating existing and emerging infectious diseases because they are short in size, easy to synthesize and proteolytically stable (Pasupuleti 2009; Seo et al. 2012).

Mechanisms of Action: How Do They Do It?

At Present several models are accounting for the antimicro- bial peptide-induced killing of microbial cells. The widely proposed and well-established mechanism of action for the destruction of microorganisms by antimicrobial peptides rely on membrane destabilization and disruption. Till date, three models of membrane permeabilization have been proposed in favor of: the Barrel–Stave, Toroidal and Carpet mod- els. Most of these are thought to exert bactericidal effects by forming pores in bacterial membranes, but their exact molecular mechanism of action remains unclear. Moreover, non-membrane or non-lytic AMPs in support with experi- mental evidence have also been described which operate through interacting with specific molecular targets (Scocchi et al. 2016).

Membrane Permeabilization Mechanism

The Barrel–Stave Model was the first proposed one, describ- ing a device in which antimicrobial peptides form a barrel- like pore in the cell membrane via inserting themselves into membrane as an individual or their complexes being the

staves spanning the membrane. The hydrophobic surface of AMPs binds with the membrane lipid core, pointing out- wards towards the acyl chains of the membrane whereas the hydrophilic surfaces pointing inward thus creating a transmembrane pore. This model has been suggested e.g. for dermcidin. In the toroidal pore model, the AMP inserts maintain a parallel orientation to the bilayer normal and interact with the membrane lipids to form a toroidal pore or worm-hole channels (Pushpanathan et al. 2013; Li et al. 2017). The membrane-spanning pore was estimated to be in size range of 2–3 nm (2 ± 0.5 nm as internal diameter and 3 ± 1 nm at the rim) (Leontiadou et al. 2006). The typical AMPs such as magainins, melittin and protegrins identified from an Afri- can clawed frog, bee venom and porcine leukocytes respec- tively were found to adapt this mode of action (Rashid et al. 2016; Li et al. 2017). Carpet model stands last in the transmembrane pore- forming mechanisms of antimicrobial peptides. They act by accumulating on the bilayer surface in a monomeric/oli- gomeric forms and reorient themselves in a manner, their hydrophobic surface of the peptides face toward the lipid side and the hydrophilic surface toward the phospholipid head groups. On attaining a threshold concentration, sur- face-oriented peptides permeate/disintegrate the bilayer in a detergent-like manner and disrupting the bilayer curvature. This model explains the activity of antimicrobial peptides such as dermaseptin, cecropin, ovispirin and aurein 1.2 (Wang et al. 2015; Shahmiri et al. 2015; Li et al. 2017). Apart from these models, molecular electroporation, sink- ing raft, interfacial activity and lipocentric pore formation models do exist, providing insight about interaction of small, unstructured and compact peptides with lipid bilayer, which lack membrane spanning capacity to form a pore directly (Hamoen and Wenzel 2017). Latest reports of hexapeptide (RWRWRW-NH2) and lipopeptide antibiotic daptomycin have strengthened the fact that many lytic AMPs are non pore formers; rather induce bacterial lysis by inhibiting cell wall synthesis and interacting with fluid membrane micro- domains thus delocalizing phospholipid architecture (Hen- derson et al. 2016; Schmitt et al. 2016a, b). In order to thoroughly understand the mode of action of AMPs and subsequent cellular events, characterization of these molecular processes is very important (Lee et al. 2015). AMPs -membrane interaction, destabilization and damage is the crucial play in the destruction of microbes. Optical biosensor technology has enabled characterizing membrane interactions by quantitation of binding events (Hirst et al. 2016). Surface plasmon resonance (SPR) is the most commonly used optical biosensor technology employed vastly for investigating membrane-mediated events. At present, a number of advanced and promising biosensors such as dual polarisation interferometry (DPI),

International Journal of Peptide Research and Therapeutics

plasmon waveguide resonance spectroscopy (PWR) and optical waveguide light mode spectroscopy (OWLS)have been developed, which provides an advantage of simultane- ous measurement of structural changes and mass binding thus facilitating to observe membrane structure changes dur-

ing peptide and protein binding (Lee et al. 2015; Hirst et al. 2016; Jirku et al. 2015).

A recent study using DPI have provide a new insight into

the membrane-penetrating property of penetratin and R8K- biotin on membrane bilayer structure during binding and insertion. Three distinct states namely a labile surface-bound state, securely bound intermediate and membrane disrup- tion following translocation were observed and the kinetic parameters were also investigated (Jirku et al. 2015). In a similar study the correlation between structural domain/ motif and membrane-binding characteristics of maculatin- related peptides was seen (Sani et al. 2015). Thus helping to develop deeper understanding about mechanisms of anti- microbial peptides (AMPs) and design of novel and highly selective antibacterial drugs (Sani et al. 2015; Choi et al.

2016).

Non‑membrane Permeabilizing Modes of Action

There are widespread shreds of evidence indicate that anti- microbial peptides, apart from their membrane-permeabi- lizing mode of action, also operate through interacting with

intracellular targets. Examples of the intracellular activity include, inhibition of DNA and protein synthesis, interfer- ing with protein folding and key metabolic enzymes, and inhibition of cell wall synthesis and septum formation. The non-lytic and non-membrane permeabilizing peptides with specificity over molecular targets have attracted substantial attention as promising antibiotics for therapeutic applica- tions and disease control (Bechinger and Gorr 2016). Repre- sentative examples include Polyphemusin (horseshoe crab), Pleurocidin (winter flounder) and Tritrpticin (mammalian bone marrow). The following will brief on less documented internally acting AMPs (Tables 1, 2, 3, 4 and 5).

In 2016, Mario et al. reported on the killing mechanism

of Bac7 135 , a novel proline-rich AMP of cathelicidin fam- ily found to selectively and comprehensively inhibiting protein synthesis by interacting with 70 s ribosome and chaperone proteins (Mardirossian et al. 2014). Further, insect-derived Drosocin, Oncocin, Pyrrhocoricin, Apidae- cin and the cathelicidin-like Bactenecin and PR-39 from mammals follow the same mechanism to inhibit bacte- rial protein synthesis (Mardirossian et al. 2014; Limoli et al. 2014). Further, were A human cathelicidin LL-37 was found to interact with DNA and disrupts normal DNA replication thereby inducing mucA mutations in P. aerugi- nosa, which is a key for phenotypic change from a non- mucoid to a mucoid appearance, which directly correlates

with worsening of clinical prognosis and cause of death in CF patients (Limoli et al. 2014). Similarly, LBP6A dem- onstrated interactions with DNA and DNA polymerase in vitro and was proven effective against many Gram-neg- ative bacteria, Gram-positive bacteria, and the yeast Can- dida albicans (Nam et al. 2014). Like defensins, fungal plectasin, eurocin, and copsin inhibit cell wall synthesis by binding to lipid II (Wang et al. 2015; Nam et al. 2014).

Biophysical Parameters Capable of Modulating Therapeutic Potency of AMPs

While thinking about therapeutic peptides, the fundamen- tal issues that need to be addressed are peptide potency, stability, selectivity, sustainability, and targetability (de Oliveira Dias and Franco 2015; Lomakin et al. 2015). It has now been recognized that biophysical properties such as amino acid composition, amphipathicity, hydrophobic- ity, charge, peptide sequence, pI, net charge at physiologi- cal pH, polar angles and post-translational modifications contribute significantly to the interaction and insertion of AMPs with the target cells (Wang 2012).

Peptide Sequence and Amino Acid Composition

The sequence length and composition are most important parameters determining the activity of an AMP because

a minimum of 7–8 amino acids are required to form an

amphipathic structure of a peptide molecule. The size of at least 22 amino acids is necessary for an alpha-helical AMP to adapt a Barrel–Stave model of bacterial membrane per- meabilization, while it is eight amino acids for β-sheet AMPs (Deslouches et al. 2005; Bahar and Ren 2013a, b). Berthony and his coworkers engineered LBU (12 residue peptide) by increasing its peptide length beyond two LBUs (WLBU2 peptide – 24 residue peptide) and substituted Trp residues in the hydrophobic domains displayed elevated antibacterial selectivity and saline tolerance (Deslouches

et al. 2005; Bahar and Ren 2013a, b). 18-mer peptide RI18

which is an analogue of PMAP-36 exhibited excellent activity against both bacteria and fungi, and a reduced hemolytic activity was observed in comparison to parental peptide PMAP-36 and melittin. The selectivity indexes of RI18 peptide against fungi and bacteria were improved 108 and 19-fold, respectively, compared to PMAP-36 (Lyu et al. 2016). The majority of the AMPs analyzed contain the highest content of Lys, Gly, Arg and Leu, and least of Met, Trp, His, Asp, Glu, and Tyr (Dziuba and Dziuba

2014).

International Journal of Peptide Research and Therapeutics

Table 1 A brief on structural and functional aspects of recently published peptides

Structural Class

PDB/APD ID

Name

Peptide sequence

Activity

Reference

Alpha-helix

5MMK

HYL-20

GILSSLWKKLKKIIAK

Antibacterial and

Nešuta et al. (2017)

 

antibiofilm

 

5MML

HYL-20k

GIMSSLMKKLAAHIAK

Antibacterial and

Nešuta et al. (2017)

 

antibiofilm

 

2N1C

PvHCt

FEDLPNFGHIQVKVFNHGEHIHH

Antifungal

Petit et al. (2016)

AP02895

Natto peptide

SMATPHVAGAAALILSKHPTWTNAQVRDRLES-

Anti-Gram positive

Kitagawa et al. (2017)

 

TATYLGNSFYYGK

 

AP02891

Dendropsophin 1

NLLNDALGTVNGLLGS

Anti-Gram positive

Triana-Vidal et al.

 

and anti-Gram

(2017)

negative

 

AP02883

Codesane

GMASLLAKVLPHVVKLIK

Anti-Gram positive,

Čujová (2014)

 

anti-Gram negative

and antifungal

Beta sheet containing peptides

AP02728

Sviceucin

CVWGGDCTDFLGCGTAWICV

Anti-Gram positive

Li et al. (2015]

AP02627

Ep-AMP1

CVLIGQRCDNDRGPRCCSGQGNCVPLPFLGGVCAV

Anti-Gram positive,

Aboye (2015)

 

anti-Gram negative

Peptides with over- representation of certain amino acids

AP02854

SpPR-AMP1

GYFPGRPPFPRPFPRPPSRPFPRPPFPGPFPRPYPWR

Anti-Gram positive,

Imjongjirak et al.

 

anti-Gram negative

(2017)

AP02742

ArmadillidinQ

GHLGRPYIGGGGGFN-

Anti-Gram positive,

Verdon et al. (2016)

 

RGGGFHRGGGFHRGGGFQSGGGFHRGGGFHSGGS-

anti-Gram negative

FGYR

and antifungal

 

AP02626

C. livia Cathelici- din 2

LIQRGRFGRFLGRIRRFRPRINFDIRARGSIRLG

Anti-Gram positive,

Yu et al. (2015)

 

anti-Gram negative

 

and antifungal

 

AP02601

BnPRP1 Pro-rich

PPIQNPSMAPPTQNPYGQPMTPPTQNPYGQPMAPP

Anti-Gram + and Gram −, antifungal

Cao et al. (2015)

Peptides with looped structure and single bond

AP02731

Panusin

SYVGDCGSNGGSCVSSYCPYGNRLNYFCPLGRTC-

Anti-Gram positive,

Montero-Alejo et al.

 

CRRSY

anti-Gram negative

(2017)

 

and antifungal

 

AP02605

Defensin-TK

SPAIWGCDSFLGYCRLACFAHEASVGQKECAEGMLC-

Anti-Gram positive,

Shen et al. (2014)

 

CIPNV

anti-Gram negative

 

and antifungal

 

AP02852

OsDEF7

RHCLSQSHRFKGMCVSSNNCANVCRTESFPDGECK-

Anti-Gram Positive,

Tantong et al. (2016)

 

SHGLERKCFCKKVC

anti-Gram negative

 

and antifungal

 

AP02853

OsDEF8

RTCESQSHRFKGPCARKANCASVCNTEGFPDGYCHG-

Anti-Gram positive,

Tantong et al. (2016)

 

VRRRCMCTKPCP

anti-Gram negative

 

and antifungal

Hydrophobicity and Amphipathicity

Hydrophobicity and amphipathicity are the two inevita- ble structural features that determine the overall activity of an antimicrobial peptide and contribute for the degree of peptide partitioning and self-promoting uptake across the lipid bilayer membranes respectively. Hydrophobic moment (MH) is a quantitative measure of amphipathic- ity and usually calculated as the vector sum of individual amino acid hydrophobicities, normalized to an ideal helix. An increase in hydrophobic moment correlates to increased permeabilization of the target cell membrane. The correla- tion between antimicrobial activity and toxicity resides in cell selectivity. The cationic property of most AMPs con- tributes to cell selectivity because the bacterial membrane is more negatively charged than that of a mammalian cell membrane.(Edwards et al. 2016; Liu et al. 2016; Hollmann et al. 2016) Trichogin, a short peptaibol on modifying its hydrophobicity and amphipathicity by introducing positively

charged amino-acid on the hydrophobic surface, signifi- cantly improved binding capacity and selectively in killing T67 cancer cells without affecting healthy normal cells.(Liu et al. 2016; Dalzini et al. 2016)Many structure—function relationship studies have revealed that more the peptide being amphipathic, more the hemolytic index. Hence, the critical parameter for all therapeutic AMPs, to maintain the hydrophobic—hydrophilic balance for an enhanced potency and better selectivity (Liu et al. 2016; Hollmann et al. 2016; Dalzini et al. 2016). On the other hand, AMPs with extreme hydrophobicity exhibit poorer antimicrobial activity and higher hemolytic toxicity in mammals have also been reported. Basically a lytic AMP must fulfill two major criteria’s in terms of hydro- phobicity; (i) The peptide must remain soluble in water and biological fluids to enable rapid transport and accessibility to the target microbes (low hydrophobicity is required) and (ii) Simultaneously the peptide should be able to interact with the hydrophobic region of the lipid bilayer in order to

International Journal of Peptide Research and Therapeutics

Table 2 Summary of cationic antimicrobial peptides and their mechanisms of action

Presumed activity

Peptide

Mechanism of action

References

Membrane-disruptive

Nisin, daptomycin Alamethicin Magainin, mellitin, MSI-78 Cecropin, dermaseptin, caerin, ovispirin Indolicidin Human AMP LL-37

Pore former Barrel–Stave (Helical-Bundle) model Toroidal pore (Wormhole, disk) model

Brogden (2005), Le et al. (2015) AlKhatib et al. (2014) Brogden (2005), AlKhatib et al. (2014) Brogden (2005), AlKhatib et al. (2014)

Carpet model Aggregate model carpet/toroidal pore DNA synthesis DNA synthesis, cell division

AlKhatib et al. (2014)

Membrane nondisruptive

Buforin II Indolicidin, PR39, HNP-1

AlKhatib et al. (2014) AlKhatib et al. (2014), Ingham and Moore

(2007)

Pleurocidin derivative PR39, dermaseptin

RNA, Protein Synthesis Protein synthesis, cell septum

AlKhatib et al. (2014) AlKhatib et al. (2014)

CP10A

formation

Friedrich et al. (2001), Le et al. (2016]

Dermaseptin Pyrrhocoricin, drosocin Apidaecin Pep5, daptomycin Mutacin 1140 Microcin 25, PR-39, PR-26 Histatins Coprisin Magainin 2 Papiliocin

RNA, protein Synthesis Protein synthesis Disruption of DnaK (chaperone) activity Cell wall lytic enzyme release Lipid II sequestion Cell septum formation Inhibits enzymatic activity Apoptosis-induction RecA activation in E. coli

Boman et al. (1993) Brogden (2005), AlKhatib et al. (2014) AlKhatib et al. (2014) Le et al. (2015) Müller et al. (2016) AlKhatib et al. (2014) AlKhatib et al. (2014) Müller et al. (2016) Müller et al. (2016) Müller et al. (2016)

Table 3 Antimicrobial peptides launched against infectious diseases. Source: THPdb database (http://crdd.osdd.net/raghava/thpdb/roa.php)

Name

Peptide sequence

Category

Company

Aldesleukin

MAPTSSSTKKTQLQLEHLLL Heavy chain: LIDGKMT YTSLIHSLIEESQNQQEKNE VGALAVVVWLWLWLWX

Antineoplastic agents, Anti-HIV agents Antisepsis HIV fusion inhibitors Anti-bacterial agents, anti-infective, topical, antibiotic Antiviral agents, immunologic factors, immunosuppressive agents Vaccine targeting toll-like receptor 2 Anti-bacterial agents –

Chiron Corp Eli Lilly and Company Trimeris, Roche Sanofi

Drotrecogin alfa

Enfuvirtide

Gramicidin D

Interferon alfa-n1

CDLPQTHSLGSRRTLMLLAQ

GlaxoSmithKline

OspA lipoprotein

MKKYLLGIGLILALIACKQN

SmithKline Beecham NPS Pharmaceuticals SciClone Pharmaceuticals (SCLN)

Teicoplanin

Thymalfasin

SDAAVDTSSEITTKDLKEKK

perturbate and delocalize the membrane architecture high hydrophobicity is required (Farkas et al. 2017). Peng et al. (2017), synthesized four AMPs based on the sequence of aa 113–145 of LysAB2 and investigated the modulations in antimicrobial potency while varying hydro- phobicity level and cationic charge distribution. A peptide variant, LysAB2 P3 (net charge: + 6.0, hydrophobic ratio:

42%) displayed 16 folds higher antibacterial activity against A. baumannii with very little haemolytic and no cytotoxic activity against normal eukaryotic cells as compared to the

parent AMP LysAB2 (net charge: + 4, hydrophobic ratio:

45%). Hence it can be speculated that optimized peptide hydrophobicity and charge distribution promotes efficient antimicrobial activity. Apart from hydrophobicity, the net positive charge of an AMP is also essential for binding with microbial membranes, thereby initiating peptide-membrane interactions. In a similar study by Yin et al. the authors detail that the phenomenal difference in antimicrobial activity and toxic- ity while altering hydrophobicity might be due to charge

International Journal of Peptide Research and Therapeutics

Table 4 Status of recently designed peptidomimetic molecules. Source: Data obtained from DRAMP database http://dramp.cpu-bioinfor.org/ browse/ClinicalTrialsData.php

Peptide Name

Sequence

Stage of Development

Description

RDP58 (delmitide)

H2N-D-Arg-D-Nle-D-Nle-D-Nle-D-

Phase II (completed)

Semisynthetic D-amino acid decapeptide derived from HLA class I B2702

Arg-D-Nle-D-Nle-D-Nle-Gly-D-

Tyr-NH2

PMX-30063 (brilacidin)

Phase II

Defensin structural mimetic, non-peptide, small molecule/copolymer Synthetic cationic host defense peptide(22-mer), magainin derivative Synthetic 12-mer cationic peptide derived from indolicidin Synthetic 24-mer peptide derived from LL-37 for binding to lipopolysaccharides or lipoteichoic acid Synthetic compound comprising fatty acid and lysine copolymers Synthetic derivative from HDP α-melanocyte- stimulating hormone Synthetic chimeric 37-mer derived from combi- nation of two natriuretic peptides Derivative of IDR-1 and indolicidin Derivative of bactericidal/permeability-increasing protein 9-amino-acid peptide derivative of bactericidal/ permeability-increasing protein HSP60 derivative (24-mer peptide) that induces T regulatory cells

Pexiganan acetate [MSI-78]

GIGKFLKKAKKFGKAFVKILKK

Phase III (faliure)

Omiganan (CLS001)

ILRWPWWPWRRK

Phase II

OP-145 (24-mer peptide)

IGKEFKRIVERIKRFLRELVRPLR

Phase II (completed)

BL2060

Lead optimization

AP-214

Phase II (completed)

CD-NP

Phase II

IMX942

KSRIVPAIPVSLL

Phase II

Opebacan

Phase I/II

XOMA-629

Phase IIA

DiaPep277

Phase III

neutralization effect. This happens because of dehydration experienced by the membrane bound AMP, which results in formation of β-type aggregates at the membrane surface and possibly leading to precipitation - thereby limiting the peptide concentration impacting on the bacterial membrane, and consequently reducing antimicrobial activity (Yin et al. 2012). Besides Hydrophobicity and cationic charge, the pep- tide length is another critical factor determining the efficacy. According to the Barrel–Stave model the AMPs penetrate perpendicular into the membrane. Hence the length of an AMP must be greater than or equivalent to the thickness of the target’s membrane (Yin et al. 2012; Yu et al. 2017).

Charge (Q) and Polar Angle (θ)

Despite the structural and compositional diversity, most of the AMPs characterized till date possess an overall positive charge ranging from + 2 to + 9 along with highly defined cationic domain(s). Cationicity is certainly an important factor for an initial electrostatic attraction of AMPs towards the negatively charged head groups of the bacterial phos- pholipid bilayer and other microorganisms. In bacteria, the presence of acidic phospholipids (PG, PS, and CL) in the cell membrane contributes for its net negative charge. Fur- ther, the LPS and teichoic acid present in Gram-positive

and Gram-negative bacterial membrane, contribute for an additional negative charge. This results in nearly 50% greater transmembrane electrical potential in prokaryotes in com- parison with most mammalian cells (Wang et al. 2015; Liu et al. 2015; Yeaman and Yount 2003). The above-mentioned facts define the strong correlation between peptide cationicity and antimicrobial activity, and this has been validated in many studies. Studies with cati- onicity enhanced analogues of the antimicrobial peptides (AcrAP1 and AcrAP2), from the scorpion venom, exhib- ited a broader spectrum of antimicrobial activity with an enhanced potency. In contrast, the native peptides expressed a narrow spectrum of activity with moderate potency. The peptide analogues modified to improve biological potency and spectrum of action surprisingly displayed growth modu- lation effects on a variety of human cancer cell lines (Liu et al. 2015; Yeaman and Yount 2003). Polar angle is a measure of the relative proportion of the polar and nonpolar facets present in an amphipathic α-helical peptide. This parameter enables to understand the distribution of hydrophobic and hydrophilic amino acid in a helical peptide. As an example, consider a α-helical peptide, which is exclusively composed of hydrophobic amino acid residues on one side and the other side solely composed of charged amino acids, then

International Journal of Peptide Research and Therapeutics

Table 5 List of latest peptides with their medical application. Source: Data obtained from DRAMP database http://dramp.cpu-bioinfor.org/ browse/ClinicalTrialsData.php

Peptide Name

Sequence

Developmental stage

Medical Use

Colicin E1 (bacteriocin)

Preclinical

Gastrointestinal tract infections; Stomach and intestine infections Urogenital tract infections; Bacterial vaginosis Gastrointestinal tract infections; Campy- lobacter infection

Lactocin 160 (bacteriocin)

Preclinical

Bacteriocin OR-7

KTYYGTNGVHCTKNSLWGKVR-

Preclinical

LKNMKYDQNTTYMGRLQDILLG-

WATGAFGKTFH

Pediocin PA-1 (bacteriocin)

KYYGNGVTCGKHSCSVDWG-

Preclinical

Gastrointestinal tract infections; Stomach and intestine infections

KATTCIINNGAMAWATGGHQGN-

HKC

Nisin A (Type A LANTIBIOTIC)

ITSISLCTPGCKTGALMGCNMK-

Preclinical

Urogenital tract infections; Spermicidal activity Staphylococus aureusanti-infectives Staphylococus aureusanti-infectives Staphylococus aureusanti-infectives Staphylococus aureusanti-infectives Staphylococus aureusanti-infectives Gram-negative infections Nosocomial infections, febrile, neutro- penia Gastrointestinal tract infections; Stomach and intestine infections (Rat model; In vivo) Hospital-acquired infections; Multi-drug resistant strain (Murine model; In vivo) Gastrointestinal tract infections; Stomach and intestine infections (Human model; In vivo)

TATCHCSIHVSK

Bac8c

RIWVIWRR

Preclinical

Temporin10a

FLPLASLFSRLL

Preclinical

Syphaxin(SPX1-22)

GVLDILKGAAKDLAGHVATKVINKI

Phase I

IDR-1002

VQRWLIVWRIRK

Preclinical

Buforin II

TRSSRAGLQWPVGRVHRLLRK

Preclinical

SB006 (M6)

QKKIRVRLSA

Preclinical

IMX942

KSRIVPAIPVSLL

Phase II

Ruminococcin C (bacteriocin)

Preclinical

Planosporicin (bacteriocin)

Preclinical

ESL5

Preclinical

the polar angle will be 180° (Tseng et al. 2016). Further, an imbalance between these proportions or a decrease in the hydrophobic ratio in helix composition will pro- portionately increase the polar angle and vice versa. There is an inverse correlation between the polar angle and membrane permeabilization capacity of the peptide (Tseng et al. 2016; Duong et al. 2016). Usually, peptides with narrow polar angles (having greater hydropho- bic surface) can easily destabilize cell membrane lipid structure and often require a smaller quantity of peptide molecules and a large number of lipid molecules as com- pared with peptides having a wide polar angle. The cor- relation among polar angle, peptide stability and half-life of peptide-induced membrane destabilization have also been investigated and reported in many studies (Tian et al. 2016; Polanco 2013; Ebenhan et al. 2014). However, the transmembrane pores induced by the smaller polar angles peptides were less stable compared to those formed by peptides with a wider polar angle (Kumar et al. 2016).

Challenges Involved in Use of AMPs as Therapeutics

Pharmaceutical and healthcare industries today, view AMPs as a promising class of therapeutic molecules to combat antimicrobial-resistant bacterial pathogens. The remarkable development of peptide therapeutics in the last decade led to a surprising number of marketing approv- als in the year 2012 and has been reported that more than 100 peptide drug candidates were still in the pipeline for approval. Peptides offer certain advantages as drugs; which include high biological activity, specificity and low levels of toxicity. However, challenges also exist for the development and use of the peptide as therapeutics. Some notable obstacles are; systemic route for administration, stability, in vivo kinetic parameters, toxicity, permeability and so on (Hutchings et al. 2017).

International Journal of Peptide Research and Therapeutics

The Right Peptide Form

The therapeutic efficacy of AMPs in vivo is often over-

shadowed by their inability to reach the targets in an active form. The physicochemical properties of a peptide greatly influence its pharmacokinetic profile and metabolic fate

in the human system. Chemical degradation pathways for

peptides and proteins such as deamidation, racemization, isomerization, hydrolysis, disulfide formation/exchange, β-elimination, and oxidation are the hindrances to over- come (Manabe and Kawasaki 2017). Different strategies are being developed and implemented to improve the stability

and functionality of peptide drugs. In specific, the suscep- tibility of AMPs to proteases in vivo is a major challenge that limits their application in pharmaceutical industry. In order to overcome this limitation, now the AMPs are being synthesized as d -amino acids instead of l -amino acids, which make the AMPs to withstand proteolytic degrada- tion. Recently, Manabe and Kawasaki, have investigated the antimicrobial properties of d and l -forms of sapesin

B (KLKLLLLLKLK-NH2). From the results, it has been

observed that d -form expressed higher antimicrobial activ-

ity against bacterial pathogens S. aureus and E. coli, relative

to its l -form. Moreover, it has also been noticed that the

elevated antimicrobial effect of d-form was not because of its resistance against proteolytic degradation and instead due to

its membrane specific interactions made along with bacterial

cell surface components (Zhao et al. 2016). In another study using Polybia-MPI a cationic peptide, the substitution with d-lysines and the d-enantiomers for all of its l-lysines greatly improved its stability against protease and a significant anti- microbial activity was also observed in comparison to its l -counterpart. Interestingly, the d -amino acid substitution turned right-hand α-helical conformation of the peptide to left-hand conformation and also further decreased its hemo- lytic activity (Afacan et al. 2012).

Manufacturing and Cost Considerations

Though antimicrobial peptides are promising alternative to traditional antibiotics, their utility is greatly limited due to its high production costs involved, and thus the large-scale production remains a challenge. To overcome this impedi- ment, an effective strategy needs to be devised. The cost per peptide could be minimized through adapting low-cost plat- form technology and by bringing in improvements in process strategies. Unfortunately, the current practice of solid-phase peptide synthesis is prohibitively expensive and requires expensive chemical precursor components during its large- scale production. Although several approaches have been proposed for industrial production of AMPs, till date, scale- up has not been proven cost-effective. Even if production is possible, further biochemical steps to facilitate purification

are quite expensive and significantly reduce yield (Mar- tin et al. 2015). In contrast to fluorenylmethoxycarbonyl (FMOC) and other chemical syntheses of peptides, which are complex and costly, recombinant platform offers cost- effective approaches for the large-scale production. On the other hand, the inherent complexity in using bacterial cell as AMPs synthesizing factories has to be overcome (Zhao et al. 2015, New antimicrobial peptide kills strains resistant to existing antibiotics 2016; Silva et al. 2016). Among the recombinant studies reported for the production of AMPs in microbial systems, E. coli host has been widely employed. The major difficulties involved in the production of AMPs in host systems include proteolytic degradation of peptides and intrinsic antimicrobial activity exerted to the host sys- tem by AMPs during its expression. At present, AMPs are fused to protein carriers such as solubility enhancing carri- ers, aggregation-promoting carriers, self-cleavable proteins and signal secretion proteins. Thioredoxin and glutathione transferase (GST), were the most frequently reported car- rier proteins which account for more than one-third of all reported fusion expressions. Whatever the technology might be, all the methods need to rely on expensive chromatog- raphy-based purification techniques to purify antimicrobial peptides, which stands as a major setback in industrial scale production of AMPs (Liu et al. 2017; Bommarius et al.

2010).

A number of researchers were attempting to address these issues. Novozymes, a Danish biotech company, has developed a novel antimicrobial peptide variant (Plectasin NZ2114) which employs high-yield fungal expression sys- tem for large-scale production of this peptide in a highly pure state (Lee et al. 2016; Mathur et al. 2016). Recently, a mass production strategy involving fusion protein technology was studied for high yield production of certain antimicrobial and immunomodulatory peptides (IDRs) including LL37, CRAMP, and HHC-10. Here in this method, an intrinsic cleavage system is utilized for freeing the pure peptide from its fusion partner and in a simple two-step process, high level of purification is achieved. The peptide was cleaved from its fusion partner using a unique enzyme sumoase (Premdjee and Payne 2017). Unlike classical chemical cleav- age approaches such as Cyanogen bromide, which cleaves C-terminally after methionine and hydrochloric acid based cleavage of the acid-labile peptide bond between asparagi- nes and proline (Schneider et al. 2010; Arenas et al. 2016). Sumoase protease Ulp1 follows a unique mechanism by rec- ognizing and specifically cleaving Gly–Gly residues after the C-terminal present within SUMO, thereby releasing AMP sequence, leaving no unwanted amino acids at the N-ter- minus of the peptide. This may be a promising technology for cost-effective industrial-scale production of AMPs and IDRs (Bommarius et al. 2010; Premdjee and Payne 2017; Mathur et al. 2016).

International Journal of Peptide Research and Therapeutics

In vivo Toxicity and Stability

Therapeutic peptides are often characterized by their high biological activity, high specificity and low toxicity (Less accumulation in organs, Low or no hemolysis and drug–drug interaction) (Gentilucci et al. 2010). Despite numerous advantages, challenges include high production cost, low storage stability and suboptimal in vivo half-life (Jenssen and Aspmo 2008).Metabolic stability of peptides is the greatest obstacle faced on the way toward successful devel- opment of bioactive peptides into therapeutic drugs in the market. Methods are being developed to stabilize peptides without reducing their effectiveness and affinity towards the target molecule. Macrocyclic peptides or polycyclic peptides are classical approaches to protect the peptides against metabolism or to prolong their half-life. A variety of other methods is still being explored and reported toward protecting peptides against metabolism or to extend their half-life. The approaches include derivatizing peptides with carbohydrates (glycopeptides), incorporation of nonnatural amino acids, which are not easily recognized by hydrolytic enzymes and by introducing structural modifications to sterically encumber the labile sites (Gentilucci et al. 2010; Jenssen and Aspmo 2008). In recent days, a growing inter- est in selective derivatizing of peptides with carbohydrates has been observed, since glycosylation can confer superior therapeutic efficacy, increased stability, higher aqueous solu- bility, better bioavailability, enhanced target resolution and longer in vivo half-lives. Since toxicity being one of the bot- tlenecks in peptide-based therapy, at present in silico models are also being developed for predicting toxicity of peptides and proteins. A peptidomimetic is one of the leading and well-estab- lished approaches in biotechnology and nanotechnology which could be adapted for developing novel, effective non- toxic therapeutic agents for performing specific operations within a physiological environment (Gentilucci et al. 2010; Ngambenjawong et al. 2016). Another major reason behind peptide stability is rapid renal clearance, and this is also due to protease degradation which results in low in vivo bio- availability, short half-life and limited access to intracellular space. In case of synthetic peptides, conformational stabil- ity is a debatable thing (Nordström and Malmsten 2017). For successful development of peptide drugs, preliminary screening assays are most important and essential for the elimination of unstable peptides entering into drug devel- opment pipeline, which considerably affects the production cost and efficiency. Stability of peptide in serum can be determined using techniques such as reverse phase–high- performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS) approach for both in vitro and in vivo (Ngambenjawong et al. 2016; Nordström and Malmsten 2017; Weinstock et al. 2012).

Incorporation of unnaturalamino acids, peptide trunca- tiona and inducing chemical modifications could be some of the possible ways to reduce invivo toxicity and improve invivo peptide stability. Koh et al., has demonstrated enhanced antimicrobial activity and stability upon intro- ducing lipid modification. It should also be noted that the modification should not disturb the cationic and hydropho- bic balance (Li et al. 2017; Koh et al. 2015). Recently a new class of peptidomimetics termed “AApeptides”, which are oligomers of N-Acylated-N-Aminoethyl amino acid has been developed which mimic the action of Host defense pep- tides capable of inhibiting the growth of multidrug resistant Gram-positive and Gram-negative bacteria. These peptides are protease resistant since it has been built on non-natural structural backbone. α-AApeptides and γ-AApeptides are two major sub-types of AApeptides (Sang et al. 2017).

Delivery Platforms: Barriers and Challenges

Delivery systems play a vital role in the development of potent and safe AMP-based therapeutic drugs. The major objectives of delivery systems designed for peptide thera- peutics include (i) Preservation of chemical or biological activity of AMPs either in the formulation or after admin- istration, (ii) Reduced toxicity and adverse side effects, (iii) sustained and or controlled release rate of AMPs, (iv) pro- mote biofilm penetration, or co-localization with intracel- lular pathogens (Kovalainen et al. 2015; Thota et al. 2016). In recent years, a variety of advancements has been made in peptide drug delivery through active or passive transporters, carriers, transdermal and nasal delivery systems. At present, peptides are parenterally delivered (via injection), since oral administration leads to inactivation and degradation in the digestive tract. The other obstacles include short half-life, lower penetration capacity and targeted delivery (Elofsson et al. 2015). Various nanoformulation platforms for targeted peptide delivery, controlled and or sustained-release strate- gies and technologies offering proteolytic stability are being investigated (Lee and Lee 2015; Widenbring et al. 2014; Muheem et al. 2016). Microgels and related polymeric systems offer exciting opportunities for delivery of AMPs and low-Mw drugs (Fosgerau and Hoffmann 2015). Fur- thermore, from industrial perspective, design simplicity, robustness, and scale-up approaches are likely to be the key for any peptide delivery systems (Kovalainen et al. 2015; Ghosh 2016).

Global Market for Therapeutic peptides

Over the past decade, therapeutic peptides have gained a wide range of applications in the fields of healthcare and Biopharmaceuticals. Furthermore, in recent days the

International Journal of Peptide Research and Therapeutics

research on therapeutic peptide is undergoing a renaissance for commercial reasons. Novel strategies in peptide design have propelled a wave of peptide therapeutics in market and research. Currently, an approximate of 140 therapeutic pep- tides are in clinical trials, and over 500 were in preclinical development (Fosgerau and Hoffmann 2015). The commer- cial market for novel peptide therapeutics was estimated to be $17.50 billion in 2015. The healthcare expenditure in two of the fastest growing economies namely Brazil and India went up by 0.3 and 0.2% 2014 over 2013 respectively, which is a key economic factor driving the global peptide thera- peutics market (Bahar and Ren 2013). According to a recent report on worldwide peptide market “Peptide Therapeutics Market: Global Industry Analysis and Opportunity Assess- ment, 2015–2025”, published by Future Market Insights market intelligence and consulting firm, the global peptide therapeutics market is projected to rise over 10% CAGR during 2015–2025.

Conclusion

The rapid emergence of MDR pathogens has necessitated an urgent need to develop novel antimicrobials. As biological anti-infective agents, AMPs can be directly antimicrobial and immunomodulatory. It has been predicted that 10 mil- lion people per year will be killed by 2050 globally, causing $100 trillion loss to the global economy (Silva et al. 2016). AMPs are less likely to promote microbial resistance since

they interfere with multiple biological processes in a patho- gen or modulate the host immune system rather than target- ing the pathogen (Narayana and Chen 2015). Furthermore, AMPs are capable of suppressing harmful inflammatory responses associated with the infection caused. Peptides hold high selectivity and efficiency and, at the same time, relatively safe and well tolerated (Aoki and Ueda 2013). These properties make AMPs an attractive alternative in the treatment of MDR infections. So far thousands of natural and synthetic AMPs have been identified, reported and still being developed, but only a few of them have reached clini- cal trials successfully (Aoki and Ueda 2013). Recently, new antimicrobial peptide Clavanin-MO has been developed by

a team of researchers at MIT, University of Brasilia, and the University of British Columbia against multidrug resistant Escherichia coli and Staphylococcus aureus. This peptide is

a synthetic derivative from parent peptide Clavanin-A with

improved hydrophobicity. This novel peptide was found to suppress sepsis by stimulating production of immune media- tors GM-CSF, IFN-γ and MCP-1, thereby increasing anti- inflammatory cytokine (IL-10) synthesis and repressing the levels of pro-inflammatory cytokine (IL-12 and TNF- α). This peptide also holdsa potentiant to cure infections caused by Pseudomonas aeruginosa, which is a causative

agent for most biofilm infections and often affect the lungs of cystic fibrosis patients (105, Silva et al. 2016). In order to design and develop novel antimicrobials with desired potency and efficiency, a deeper understanding and knowl- edge over structure-activity-property–behavior relationship and relating them all together is a critical requirement for development of novel therapeutics. Riduan and co has devel- oped a novel class of antimicrobial imidazolium oligomers capable of ultrafast killing (> 99.7% killing within 30 s), and possess superior activity, excellent selectivity, self-gelling properties. The Molecular dynamic simulations studies have reavealed that ultra fast killing is due to spontaneous pene- tration and translocation across the microbial cell membrane within a very short timescale of seconds to minutes (Riduan et al. 2016). Issues regarding manufacturing cost, peptide stability, and toxicity were some of the major challenges not yet been completely addressed. Although more work is needed, sub- stantial strides have been made in the recent years to over- come these setbacks, and the future looks promising for the development and widespread use of peptides as therapeutics targeting MDR pathogens. More focus is to be made on syn- thetic or peptides mimicking natural antibiotics. Since they rarely develop resistance, this could be a possible way to fight with multi-drug resistant organisms. Only when these aspects have been unraveled in greater detail, the AMP- based therapeutics can likely to be able to reach their full potential. Above all, the best way to combat against anti- microbial resistance (AMR) is by prevention. An holistic and collaborative approach towards prevention and contain- ment of AMR across all nations, government and society is required to minimize the emergence and spread of antimi- crobial resistance. Further efforts for enhancing awareness, strengthening AMR surveillance, educating people about responsible use of antibiotics, reducing infections and pro- moting research globally is also required.

Acknowledgements The authors wish to thank SERB and UGC, Government of India, for financial support through major project. A special thanks to Prof. Dr. K. Ruckmani, Head and Director, Depart- ment of Pharmaceutical Technology, Centre for. Excellence in Nanobio Translational REsearch. (CENTRE), University College of Engineer- ing, Bharathidasan Institute of Technology Campus, Anna University, Tiruchirappalli-620024, Tamil Nadu, India for her constant support.

Funding This study was supported by Science and Engineering research board, Department of Science and Technology under the scheme of Empowerment and Equity Opportunities for Excellence in Science. Sanction Order No: SB/EMEQ-034/2014 dated 06.07.2015. University Grants Commission, National Fellowship for SC, Award Letter No.: F1-17.1/2017-18/RGNF-2017-18-SC-TAM-45554. Dated:

16/08/2017.

Compliance with Ethical Standards

Conflict of interest Authors declare no conflict of interest.

International Journal of Peptide Research and Therapeutics

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

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