Future prospects for Antimicrobial peptide development: peptidomimetics and antimicrobial combinations

1. Introduction

Antimicrobial resistance is widely recognized as one of the major challenges of public health worldwide, as infections by multidrug-resistant (MDR) bacteria have reached worrisome levels. Three levels of antibiotic resistance have been described [1]: i) multi-resistance (resistance to at least three different families of antimicrobials), ii) extreme resistance (resistance to all antimicrobials except colistin) and iii) pan-drug resistance (resistance to all available antimicrobials).

The currently high-level multi-resistance has been attributed to the global misuse and abuse of antimicrobial agents, whether in veterinary practice, the food industry, aquaculture, or medicine. Alexander Fleming, who received the Nobel prize in 1945 for his discovery of penicillin, foresaw this situation [2], and his predictions have become a very worrisome reality. Literature chronograms well demonstrate the time period marking the transition from the discovery of antimicrobials to the emergence of resistance.

With the increasing prevalence of MDR bacteria, antimicrobial peptides (AMPs) have become the focus of intense interest in the treatment of these infections. Among their advantages are their slow selection of resistant strains and their unique mechanisms of action. AMPs are highly versatile molecules that offer many possibilities for chemical modification, resulting in novel agents with improved therapeutic and safety [3]. Consequently, an enormous number of these drugs are currently in different stages of development [4,5].

Four different models have been proposed to describe the mechanisms of action of AMPs: the toroidal model, carpet model, barrel stave model, and aggregate model. In general, AMPs act by disrupting bacterial membranes, resulting in modifications of permeability and pore formation that finally lead to bacterial death. Other mechanisms of action have also been described, such as the inhibition of DNA, RNA, and protein synthesis [6], and the reduction of efflux pump activity due to the effect of AMPs on bacterial membranes [7]

Nevertheless, the disadvantages of AMPs have imposed limits on their usefulness. Chemically, the high molecular weight of AMPs complicates their synthesis, resulting in low yields and high production costs. Furthermore, the biological characteristics of AMPs can result in their cytotoxicity, instability, short half-life and rapid elimination by oxidation or proteolytic digestion, by proteases such as trypsin (hydrolysis of basic residues) and chemotrypsin (aromatic residues).

In efforts aimed at addressing these shortcomings, numerous research laboratories are attempting to develop new AMPs with improved biological characteristics.

1.1. The improved biological properties of peptidomimetics

Antimicrobial peptidomimetics are synthetic molecules derived from (or inspired by) natural AMPs that mimic or reproduce their physicochemical properties (cationic charge, hydrophobicity, amphiphilicity) and biological effects [8,9]. Nevertheless, peptidomimetics often exhibit a higher bioavailability and a longer half-life in vivo [5] and many of them are more resistant to protease degradation [10]. To date, several approaches have been used to design potent peptidomimetics with alternative backbone structures. In most cases this involves the insertion of unnatural amino acid residues, such as N-substituted glycines (peptoids), in β-peptides as well as the design of peptide-peptoid hybrids, ceragenin-based mimetics, and α/γ N-acylated N-aminoethyl peptidesin AA peptides [5].

The classes of peptidomometics described below have demonstrated improved biological properties and are thus excellent candidates for testing in combinatorial studies.

1.1.1. Peptoids

Peptoids consist of oligomers of N-substituted glycine units and comprise a new class of biologically active peptidomimetics [11]. Their main site of action is the bacterial membrane, but the precise underlying mechanism remains unclear [11]. An advantage of these molecules is their relatively long half-lives, as they are highly resistant to proteolytic degradation by trypsin and chemotrypsin and by other proteases as well [12].

Molchanova et al [13], in an extensive review on peptoids, (cyclic peptoids, peptide-peptoid hybrids, and β-peptoids), pointed out their excellent antimicrobial activity and low cytotoxicity (measured by hemolysis). These features make peptoids a promising alternative to classical antimicrobials.

Moreover, via membrane permeabilization, peptoids can potentiate the activities of other antimicrobials and thus promote the accumulation of the latter in bacterial cells, where they can exert their lethal effects.

1.1.2. AApeptides

AApeptides are oligomers of N-acylated-N-aminoethyl substituted amino acids derived from chiral peptide nucleic acid (PNA) backbones [11]. Depending on the position of the side chain they can be sub-classified into α-AApeptides and γ-AApeptides Their different structures included linear or cyclic molecules and association with a lipid chain [14].

Hybrids α/γ-AApeptides containing both α-peptides units and γ-peptide units exhibit excellent biological activity and low toxicity in terms of hemolysis. Sang et al. [14] described a series of hybrid α/γ-AApeptides with broad-spectrum antimicrobial activity. With a mechanism of action based on membrane damage, these molecules are likely to be effective when combined with other antimicrobial agents.

1.1.3. Peptides containing non-natural amino acids

AMPs made up of non-natural amino acids exhibit greater stability as they are resistant to the action of bacterial proteases.

Hicks et al. [15] studied the antimicrobial activity of a group of these AMPs against ESKAPE pathogens and demonstrated their increased potency. The authors also found that the introduced modification conferred selectivity for different membranes. This ability to determine the selectivity of AMPs has enormous advantages in terms of designing new antimicrobials with a narrow spectrum of antimicrobial activity or the inability to interact with cellular (eukaryotic) membranes, which would avoid the cellular toxicity of these compounds.

1.1.4. Stapled peptides

These peptide chains have an α-helical structure that improves their stability. Stapled peptides have been extensively examined as antitumor drugs [16] but their efficacy as antimicrobials has scarcely been investigated.

2. Combination therapy with antimicrobial peptides: old and new strategies

Although new AMPs have been improved in terms of their toxicity, resistance to proteases, and their enhancement of antimicrobial action, resistance can emerge and spread, as well demonstrated by resistance to colistin. The review by Anderson et al. [17] cited studies in which AMP-resistant bacterial mutants with cross-resistance to different AMPs were described. The authors suggested that because synthetic AMPs are derivatives of AMPs from the human innate immune system, their clinical use could select for resistant mutants that could also become resistant to the immune responses of humans [17].

Furthermore, given the rapid increase in resistance to classical antibiotics, new strategies to avoid or delay the emergence of antimicrobial resistance to these new antimicrobials, especially AMPs, are urgently needed.

It is well known that the use of antimicrobials used in combination is often an effective approach to reduce the emergence of resistance [18,19]. Several authors have suggested that combination therapy with AMPs could contribute to solving, at least in part, the problem of antimicrobial resistance [20–23].

The potential benefits of treating infectious diseases with combinations of antimicrobials are being increasingly recognized, as are the infections likely to be the most responsive to these combinations, although both remain controversial [24].

For many years, the aim of combination therapy with antimicrobial agents was to enlarge the antimicrobial spectrum of action, with a reduction to only one antimicrobial once the etiology of the infection was precisely determined. Nowadays, the main objective of antimicrobial combinations is to increase antimicrobial activity and thus overcome the mechanisms of resistance while preventing the emergence of new ones.

As noted above, AMPs increase the fluidity of bacterial membranes, thus changing their permeability and facilitating the uptake of other substances, including antimicrobials [7]. It is this synergistic ability of AMPs, rather than their intrinsic antimicrobial activity, that underlies current interest in their use in combination therapy. Further studies on the synergism between AMPs and other antimicrobials can be expected to lead to novel antibacterial drugs.

2.1. Reducing the cytotoxicity by synergism

Obtaining new AMPs with low or no toxicity is a prerequisite for the use of these agents in clinical practice. The cytotoxicity of AMPs strongly depends on their structure and composition. Gong et al. [25] reported that the hydrophobicity of AMPs is closely related to their cytotoxicity, as highly hydrophobic AMPs easily penetrate bacterial and mammalian membranes. Thus, changing the net charge, amino acid composition, length, or other structural characteristics of AMPs may improve their antimicrobial activity while reducing their cytotoxicity [25].

Since the toxicity of a drug strongly depends on its concentration, one approach to reduce or minimize the cytotoxicity of AMPs is to combine them with classical antibiotics or other AMPs. The eventual resulting synergies may allow the use of greatly reduced concentrations of both AMPs and the other components in the combination. Low concentrations of AMPs may be sufficient to disrupt cell membranes and change their permeability, thereby facilitating the entry of other antimicrobials with lethal effects limited to bacterial cells. For example, colistin, the best known and most well studied AMP, has been effectively combined with other antimicrobials, including those to which bacteria are otherwise intrinsically resistant [7].

Other AMPs have similarly shown very good results when used in combination with classical antimicrobials. Rudilla et al. [26] reported the synergy of a new synthetic colistin derivative and carbapenems in carbapenem-resistance clinical P. aeruginosa. Kumar et al. [27] examined the synergy between proline-based cyclic dipeptides of natural origin and β-lactams. In the study of Wang et al., short AMPs (PMAP- 36 and PRW4) showed synergistic effects when used in combination with aminoglycosides [28].

In summary, combining AMPs with classical antibiotics seems to be an effective approach to reduce the toxicity of AMPs, while hindering the emergence of resistant bacteria. Thus, in the development of new AMPs, their synergism with other antimicrobials should be carefully tested.

3. Expert opinion

Alerts concerning MDR bacteria have been issued by health authorities worldwide, since infections caused by these strains have increased dramatically in recent years. Alternatives to antibiotics are in the spotlight of biomedical research. One such alternative is AMPs. Consequently, intense efforts are currently underway to preserve or improve their efficacy while reducing their toxicity and maintaining their low potential for the selection of resistant strains. This research has led to the development of peptidomimetics (peptoids, AApeptides and staple peptides, families of molecules with the ability to disrupt membranes and low toxicity). The battle against multi-resistance involves different weapons to achieve the key objective of preventing the emergence and spread of antimicrobial resistance. This is likely to be achieved by i) the development of new molecules with a low potential to select resistant strains or ii) the combined use of two or more antimicrobials. In this sense, AMPs and their derivatives merit in-depth studies not only as genuine antimicrobial agents but also as enlargers of the spectrum of action of existing drugs. The use of AMPs in combination with traditional antibiotics may provide a strategy to overcome resistance arising from outer membrane permeability restrictions or drug efflux and may extend the useful life of older and new molecules. This broad field of research offers many avenues for further exploration.

Declaration of Interest

The team of M Viñas are members of the JPIAMR (Joint Programming Initiative on Antimicrobial Resistance) TRANSLOCATION-transfer network, https://www.jpiamr.eu/supportedprojects/. The research done in this lab is funded by the Marató TV3 Foundation (BARNAPA project) The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

The authors were funded by the University of Barcelona.