Modifying Natural Antimicrobial Peptides to Generate Bioinspired Antibiotics and Devices

Keywords::

• antimicrobial peptides
• bioinspired devices
• multidrug resistance

Antibacterial drug resistance is an increasingly serious threat that affects the world over and that demands our attention. Among the reasons for the observed upsurge in drug-resistant bacteria is the inappropriate usage of antibiotics in both humans and animals [1]. The emergence of antibiotic resistance has increased impetus for the development of novel potent antibiotics and as result, antimicrobial peptides (AMPs) have been identified as promising candidate molecules, due to their unusual mechanisms of action and lack of side effects.

The delicate balance of antibiotic resistance and the discovery of new antimicrobials is disproportionate, with only a small number of antibiotic molecules having reached commercial drug pipelines in the last few years [2]. In an effort to overcome this imbalance, scientists have turned to the natural environment, which has long been considered a valuable resource for the discovery of antibacterial agents. In this regard, several biomes have been explored, from the tropical rain forests to desert regions [3]. Although thousands of antimicrobials have been discovered in microorganisms, plants and animals almost few of them have as yet reached clinical trials. This is largely due to several problems hindering the drug development process such as the high structural complexity of the compounds, low antibacterial activity, compound side effects, difficulties in the production of the drug and the high costs incurred by the development process. In order to address these shortcomings, researchers have become increasingly interested in studying the genetic diversity found in many natural environments and have begun to focus on developing strategies for the improvement of the natural antimicrobial compounds. For example, there a number of cases where AMPs have been modified or grafted to generate novel bioinspired compounds against antibiotic resistance genes.

Finding the natural antimicrobial sources

Natural ecosystems provide a wide variety of unique compounds that may be explored for the creation of novel bioinspired molecules with antibiotic potential. For this purpose, intensive research efforts have been applied and sophisticated high-throughput molecular techniques have been developed and employed in the search for novel antibiotics including AMPs by using proteomics, genomics and metagenomic approaches [4], in addition to traditional isolation and bioassay platforms [5]. Traditionally, metagenomics studies have predominantly focused on discovering a vast array of antibiotic resistant genes in the natural environment [6,7]. Recently, however, metagenomic data have been used to revolutionize our knowledge of the overwhelming majority of not-yet-culturable microbial communities, and a few studies have led to the development of new potential antibiotics [8]. With the use of these high-throughput molecular approaches, it is anticipated that we will be able to gain a better knowledge of the genetic diversity present in the many different natural environments and in turn focus our efforts toward the development of new bioinspired antibiotic compounds.

Bioinspired peptides & medical devices using antimicrobial peptides

Natural AMPs are particularly promising antibacterial drug candidates as there are several manners in which we can improve their potential as a pharmaceutical. First, problems such as low activity and side effects may be circumvented by shortening the peptide to leave the sequence that is the most effective against the microbe. To achieve this, the sliding window technique may be used to screen peptides and find small sequences directly involved in the antimicrobial activities. Using the sliding window method, a constant number of amino acid residues, for example, five, may be chosen and the peptide scanned residue by residue in the direction of the N-termini. All sequences with five residues (1–5, 2–6, 3–7, etc.) may then be synthesized and tested for antimicrobial activity. Using this technique, one is able to localize the most effective antimicrobial sequence and also eliminate parts of the peptide that could act as antigens, thereby improving the immune response and reducing severe side effects. Furthermore, advanced computer-assisted design strategies could also be utilized in such an approach, generating cost-effective, potent and broad-spectrum AMPs [9].

Another common problem with antibacterial peptide development is stability. Once the peptides are injected into the host, they are rapidly destroyed by multiple extracellular proteinases. In order to avoid this issue, chimera molecules have been constructed. The chimeric AMP, cecropin A–melittin (CAM), which consists of two insect AMPs named CAM combined into a single polypeptide, serves as an example. CAM exhibits potent antimicrobial activity but is threatened by some special extracellular proteases when used to deal with certain drug-resistant pathogenic microbes in the gastro intestinal tract. To combat this issue, a four-tryptophan-substitution mutant (CAM-W) of CAM was synthesized and found to have enhanced antimicrobial activity and improved peptide proteolytic stability [10].

Should the short peptides, generated by the sliding window strategy previously described, be found to exhibit extreme susceptibility to proteolytic enzymes, the problem may be circumvented by adding several short sequences to larger and stable structural scaffolds such as the knot-fold – this is built from six conserved cysteine residues and observed in cyclotides [11]. Cyclotides are a unique family of backbone cyclized plant peptides with comparably exceptional stability to chemical, thermal and enzymatic degradation. Furthermore, the cystine knot framework is widely tolerant of a range of residue alterations, therefore holding great promise as a scaffold in drug design and protein engineering. In this regard, cyclotides present enormous potential as templates on to which AMPs may be grafted, thereby improving stability. Over the last decade, there have been numerous examples of short bioactive peptide epitopes being grafted on to cyclotides, in this way. Such a strategy has been previously used to incorporate peptides derived from MOG35–55 epitope onto a cyclotide. The study found that one of the grafted peptides, MOG3, displayed the ability to prevent against multiple sclerosis diseases in a mouse model for the disease [12].

In a study conducted by Fensterseifer and coworkers, nongrafted cyclotides, specifically cycloviolacin 2 and kalata B2, were found to demonstrate antimicrobial activity when tested against Staphylococcus aureus [13]. With this in mind, it is plausible that by grafting AMPs on to the cyclotide template, the overall bactericidal activity of the resulting molecule may be improved.

Other cysteine-rich structural scaffolds could be also used to graft short AMPs including defensins and conotoxins. In both cases, a proof of concept must be performed for developing enhanced antimicrobial activity due to the high structural complexity and the multifaceted process of producing such grafted peptides. In the latter case, complex chemical synthesis [14] or heterologous production [15] will be necessary to overcome such barriers.

Interestingly, nowadays, not only have novel bioinspired AMPs been developed but also bioinspired tools and devices. Nanofibers and modified surfaces actually using AMPs have been utilized for fighting infectious diseases. For example, the AMP lasioglossin-III (lasio-III) was covalently immobilized on a silicone catheter creating a protected biosurface. The lasio-III-coated catheter prevented Escherichia coli and Enterococcus faecalis growth [16]. Moreover, Basu et al. [17] performed the immobilization of polybia-MPI on a silicon substrate also with the aim of creating new catheters. Cutaneous antimicrobial gels have also been produced by the incorporation of AMPs such as nisin-modified hyaluronic acid (HA) polysaccharide gel, which showed antimicrobial activity against Gram-positive and negative bacteria [18]. Additionally, nanofibers have received considerable attention in biomedical technology development due to their small size and high surface area. In this context, AMPs are remarkable alternative polymers for preparing bioinspired nanofibers. For example, bacitracin has been inserted into nanofibers using self-assembly [19], leading to the construction of highly antibacterial fibers that could potentially be employed in the near future to treat burned and infected skin.

In summary, novel bioinspired AMPs and the application of AMPs in medical devices has shown great promise and I think that there is a bright future ahead. It is probable that many of the AMPs discovered in nature in the last century could not be directly used as commercial drug against resistant bacteria, but, nevertheless, the knowledge we have gained of AMPs in nature will be indispensable in the generation of novel bioinspired antimicrobials. Undoubtedly, it is necessary to keep digging deep in order to find novel compounds in the natural world in order to yield innovative tools that could be utilized to control resistant bacteria. Indeed, humanity is losing this war, and new weapons are in great demand.

Financial & competing interests disclosure

This work was supported by CNPq, CAPES, FAPDF and FUNDECT. 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.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This work was supported by CNPq, CAPES, FAPDF and FUNDECT. 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.