The value of antimicrobial peptides in the age of resistance...Maria Magana, Muthuirulan Pushpanathan, Ana L Santos, Leon Leanse, Michael Fernandez, Anastasios Ioannidis, Marc A Giulianotti,

www.thelancet.com/infection Vol 20 September 2020 e216

Review

The value of antimicrobial peptides in the age of resistanceMaria Magana, Muthuirulan Pushpanathan, Ana L Santos, Leon Leanse, Michael Fernandez, Anastasios Ioannidis, Marc A Giulianotti, Yiorgos Apidianakis, Steven Bradfute, Andrew L Ferguson, Artem Cherkasov, Mohamed N Seleem, Clemencia Pinilla, Cesar de la Fuente-Nunez, Themis Lazaridis, Tianhong Dai, Richard A Houghten, Robert E W Hancock, George P Tegos

Accelerating growth and global expansion of antimicrobial resistance has deepened the need for discovery of novel antimicrobial agents. Antimicrobial peptides have clear advantages over conventional antibiotics which include slower emergence of resistance, broad-spectrum antibiofilm activity, and the ability to favourably modulate the host immune response. Broad bacterial susceptibility to antimicrobial peptides offers an additional tool to expand knowledge about the evolution of antimicrobial resistance. Structural and functional limitations, combined with a stricter regulatory environment, have hampered the clinical translation of antimicrobial peptides as potential therapeutic agents. Existing computational and experimental tools attempt to ease the preclinical and clinical development of antimicrobial peptides as novel therapeutics. This Review identifies the benefits, challenges, and opportunities of using antimicrobial peptides against multidrug-resistant pathogens, highlights advances in the deployment of novel promising antimicrobial peptides, and underlines the needs and priorities in designing focused development strategies taking into account the most advanced tools available.

IntroductionMicrobial infections contribute substantially to global mortality trends. Although antibiotics were once capable of treating most bacterial infections, the emergence of antimicrobial resistance diminished the effectiveness of existing antibiotics. The so-called red-alert pathogens that threaten the use of most front-line antibiotics have been involved in severe bloodstream, urinary tract, respi-ratory tract, and surgical site infections.1 The design of antimicrobials that are less susceptible to evolutionary resistance mechanisms than conventional antibiotics is challenging. Bacteria possess three defined types of antimicrobial resistance: intrinsic, acquired (figure 1), and phenotypic or adaptive resis tance.2–10 Despite their known capability to elicit bacterial resistance mecha-nisms, small-molecule antimicrobials remain at the forefront of initiatives to replace antibiotics that are beginning to fail.11

Antimicrobial peptides (AMPs), a diverse group of bioactive small proteins, are part of the body’s first line of defence for pathogen inactivation. They work by disrupting bacterial cell membranes, modulating the immune response, and regulating inflammation.12 AMP reservoirs and expansion beyond the available chemical space remains high13 because there is a plethora of un-conventional sources of AMPs, including unculturable soil and marine bacteria, and methods available to produce vast libraries of derivatives. Besides the use of AMPs in treating infections, they can also be applied to control plant diseases. Synthetic analogues of natural AMPs have been expressed in transgenic plants to confer protection against infection or are secreted by microorganisms and are then used as active ingredients of commercial bio-pesticides.14 Recombinant or synthetic AMPs can be safely used as therapeutics in aquaculture, food additives for livestock, or food preservatives.15,16 This Review outlines the current status of AMP research and development strategies, placing discoveries and methodologies in context, providing a snapshot of bacterial resistance

mechanisms (panel), and bridging the gap between disco-very and clinical application. This information is valuable in identifying the real potential of AMPs in antimicrobial drug design and development.

AMPs and natural host immunityAfter Alexander Fleming’s discovery of the lysozyme in 1922, it was not until the mid-1990s that the first AMPs were identified after Drosophila melanogaster bacterial or fungal infection.24 The 3D structures of defensins in Drosophila are similar to human β-defensins, suggesting a common origin, whereas the remaining Drosophila AMPs share homologues only with insects.25 Thus, the evolution of AMPs in insects and other species indicates their potential for antimicrobial specificity, which can be exploited in antimicrobial discovery. Differences within

Lancet Infect Dis 2020; 20: e216–30

Published Online July 9, 2020 https://doi.org/10.1016/ S1473-3099(20)30327-3

Department of Biopathology and Clinical Microbiology, Aeginition Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece (M Magana MSc); Department of Human Evolutionary Biology, Harvard University, Cambridge, MA, USA (M Pushpanathan PhD); Department of Chemistry, Rice University, Houston, TX, USA (A L Santos PhD); Investigación Sanitaria de las Islas Baleares, Palma, Spain (A L Santos);

Department of Dermatology, Harvard Medical School, Boston, MA, USA (L Leanse PhD, T Dai PhD); Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA (L Leanse, T Dai); Department of Urologic Sciences, Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada (M Fernandez PhD, Prof A Cherkasov PhD); Department of Nursing, University of Peloponnese, Sparta, Greece (A Ioannidis MD); Torrey Pines Institute for Molecular Studies, Port St Lucie, FL, USA (M A Giulianotti PhD, C Pinilla PhD, R A Houghten PhD); Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus (Y Apidianakis PhD);

Department of Internal Medicine, Center for Global Health, University of New Mexico, Albuquerque, NM, USA (S Bradfute PhD); Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, USA

(A L Ferguson PhD); Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, IN, USA (Prof M N Seleem PhD); Machine Biology Group,

Key messages

• The conformational and physicochemical properties of AMPs play important roles in determining antibacterial activity, toxicity, and bioavailability

• AMPs favour topical over systemic administration• Previous work has produced at least 2800 AMP molecules

with only a few antibacterial candidates advancing to preclinical and clinical development

• Broadening the spectrum of mapped chemical space is a promising solution against antimicrobial resistance

• Interdisciplinary expertise is necessary to discover and advance new antimicrobial agents to clinical use

• Validation of drug discovery approaches will be enhanced by more AMPs moving into phase 3 clinical trials

• Nanotechnology has entered the realm of antimicrobial drug discovery and it will accelerate the performance of intelligent drug-delivery systems with the purpose of maximising therapeutic effects and minimising undesirable side-effects.

AMP=antimicrobial peptide.

http://crossmark.crossref.org/dialog/?doi=10.1016/S1473-3099(20)30327-3&domain=pdf

e217 www.thelancet.com/infection Vol 20 September 2020

Review

Departments of Psychiatry and Microbiology, Institute for

Biomedical Informatics, Institute for Translational

Medicine and Therapeutics, Perelman School of Medicine,

Penn Institute for Computational Science, and

Department of Bioengineering, University of Pennsylvania,

Philadelphia, PA, USA (C de la Fuente-Nunez PhD);

Department of Chemistry, The City College of New York, New

York, NY, USA (Prof T Lazaridis PhD); Graduate

Programs in Chemistry, Biochemistry, and Physics,

The Graduate Center, City University of New York, NY,

USA (Prof T Lazaridis); Department of Microbiology and Immunology, Centre for

Microbial Diseases and Immunity Research, University

of British Columbia, Vancouver, BC, Canada

(Prof R E W Hancock PhD); Reading Hospital, Tower

Health, West Reading, PA, USA (G P Tegos PhD); and

Micromoria, Venture X Marlborough, Marlborough,

MA, USA (G P Tegos)

Correspondence to: Dr George P Tegos, Reading

Hospital, Tower Health, West Reading, PA 19611, USA

[email protected] Drosophila genus are subtle and can be explained by convergent balancing selection, according to which combinations of different structural classes and variants of AMPs are selected to fine-tune host defenses.26 This finding indicates that AMPs vary in their effectiveness to specific microbes, but also act synergistically. Evolution might be guided by another property of AMPs—interaction with host cells to modulate the immune response,27 which in turn could have consequences for organism health.28 On one hand, administration of bacterial AMPs through feeding resulted in toxicity and contributed to polyQ-mediated neurodegeneration in Drosophila.29,30 On the other hand, diptericin increased the lifespan of flies during hyperoxia by inducing an anti-oxidant glutathione S transferase-related response.31

As with most drugs, the dose, duration, route of application, formulation, target tissue, and host microbiota dictate the effectiveness of AMPs. Particularily, intestinal microbiota control of AMP expression reduces infla-mmation and sustains bacterial colonisation.32 Autophagy modu lation has anti-inflammatory effects and reduces the microbial burden by targeting intracellular pathogens.33 In a bidirectional relationship between peptides and host immune factors, AMPs can act as chemoattractants that initiate the recruitment of innate immune cells to the infection site and initiate chemokine induction by various

cells.34 In some chronic autoimmune diseases, such as systemic lupus erythematosus or scleroderma, cytokine-induced cathelicidin expression and over expression of human defensins confers protection against skin infec-tions,35 whereas the anti-inflammatory nature of AMPs can suppress both chronic and acute inflammatory con-ditions.34,36 AMP secretion following Toll-like receptor stimulation occurs in psoriasis, rosacea, and diabetic foot ulcers, whereas in atopic dermatitis, interleukins drive the suppression of peptide expression.37,38 In the gastrointestinal tract, Paneth cells trigger bacterial recognition and anti-microbial activity through MyD88-dependent Toll-like receptor activation.39 The secretion of human defensins following activation of mitogen-activated protein kinase and Janus kinase-signal transducer and activator of trans-cription (JAK/STAT) signalling pathways confers luminal protection from non-commensal gut pathogens including Clostridioides difficile and Helicobacter pylori.40,41 Mapping the diversity and phylogenetic distribution would enable pre-diction of the evolutionary origins of some AMP families (figure 2), identify con served and taxon-specific families, examine alternatives for conventional antibiotics to restore the susceptibility of multidrug-resistant pathogens, and facilitate development of templates for the rational design of peptidomimetic drugs.42 Moreover, the mapping could guide the identification of multiple potent combinations to

Figure 1: Mechanisms of antimicrobial action and resistance development by Gram-negative bacteriaLeft section (indicated by the dashed yellow line) of the bacterial cell shows the mechanisms of action of AMPs. Right section of the bacterial cell shows AMP resistance mechanisms in Gram-negative bacteria. AMP=antimicrobial peptide.

β-lactamases

Membranepermeabilising

Non-membranepermeabilising

Translation

Cell wallsynthesis

DNAreplication

Transmembrane efflux pump

Antibiotic

Plasmid withantibiotic-resistantgenes

Antibioticmodifyingenzyme

Modification inlipopolysaccharide

Target mutations (DNA gyraseand topoisomerase IV)

Transcription

Ribosomalmutation ormodification

Metabolic genesand regulators

Changes in membranepermeability

Antibioticinactivation

Target modifications

Loss of porins


Review

alleviate antimicrobial resistance. One potential application would then be the administration of inducing chemicals that could enhance the production of natural peptides.43

Antimicrobial resistance exhausts therapeutic optionsSoil, human, and marine microbiota harbour bacteria that compete for nutrients and space. This competition involves the synthesis and continuous evolution of new AMPs, and therefore might harbour an untapped reservoir of potentially relevant antimicrobial products.44 Efforts in antimicrobial discovery have been diverted towards unexplored and unique environments (extreme temperature, pressure, salt, pH). For example, thermo-actinoamide A is a peptide produced by microorganisms thriving under extreme natural environments, which could be promising in the search for new antibiotic agents.45 This great plate count anomaly detected in oligotrophic, mesotrophic, and eutrophic marine eco-systems denotes the limitations of conventional micro-bial cultivation techniques.46 The great plate count anomaly refers to most microbes seen under the micro-scope that cannot be cultivated in standard laboratory conditions; some might be nonviable, whereas others are viable but non-culturable. On the basis of this obser-vation, the unculturable organisms represent a pool of unexplored biodiversity that sparked the develop ment of new cultivation strategies. Eleftheria terrae was previously

an unculturable bacterium that was found to carry an AMP of high medicinal value—teixobactin. This peptide-like secondary metabolite showed activity against Gram-positive pathogens and Mycobacterium tuberculosis. Since teixobactin specifically targets con served substrates (lipid II and III) rather than enzymes in cell wall bios-ynthetic pathways, it is unlikely for bacteria to develop resistance.47 Certain AMPs share the same mechanism and have a decreased propensity to induce resistance.48,49

In early 2018, to assess natural products with anti-microbial activity, scientists used a culture-independent discovery platform, involving DNA extracted from environmental samples, which led to the discovery of malacidin. Malacidin (metagenomic acidic lipopeptide antibiotic) also targets lipid II, is non-toxic to humans, and shows reduced susceptibility to generate resistance.50 Teixobactin and malacidin are potent drug candidates, lethal against methicillin-resistant Staphylococcus aureus (MRSA) without traceable resistance development, and both have low cytotoxicity. Gram-positive actinomycetes have long been investigated for their capacity to produce

Panel: Mechanisms of bacterial resistance to AMPs

There are four main mechanisms of resistance to AMPs: (1) proteolytic degradation of peptides, (2) target alteration through bacterial surface modification, (3) shielding of the bacterial membrane by the production of a capsule, and (4) down-regulation by immunomodulation.17 Analysing the bacterial resistome reveals genetic markers related to cell surface modification, efflux pump activation, and AMP degradation.18 Physicochemical alterations in membrane rigidity, thickness, and charge confer resistance affecting surface attachment.19 Multidrug efflux systems in bacterial pathogens share the biological complexity, overlapping specificity, and evolutionary adaptability to antimicrobials.20 The resistance pattern of the slimy anionic biofilm matrix restricts AMP effectiveness by delaying diffusion into the bacterial aggregate and inducing conformational changes.21 Escaping resistance explains why membrane permeabilisers are preferred over AMPs that target intracellular proteins. Synthetic chemical pathways enable the design of peptides interacting with the bacterial cell membrane without requiring genetically driven target affinity.22 Single AMPs that target the bacterial cell membrane and an intracellular target, or multiple AMPs synergistically acting against a single bacterial target might also provide alternative ways to combat the global increase in antibiotic resistance.23

AMP=antimicrobial peptide.

Figure 2: The evolutionary relationship of AMPs effective against Gram-negative bacteriaA total of 38 AMP sequences were retrieved from The Antimicrobial Peptide Database on the basis of their potential antimicrobial activity against Gram-negative bacteria. This phylogenetic tree was developed using the maximum likelihood method on the basis of AMP sequence alignment, made with the ClustalW algorithm in MEGA 5.2 software. AMPs on external nodes (tips) of the phylogenetic tree show the derived AMPs and AMPs at internal nodes represent hypothetical ancestors. AMP=antimicrobial peptide. LAP=lingual antimicrobial peptide.

PR-39

BAC5 (cathelicidin-2)

Brevinin 1

TRan

alexin

Aurein

Caeri

nHep

icidinForm

aecin

Escu

lent

in 1

Rana

teur

in 1

Pone

ricin

Max

imin

Pleu

rocid

inDr

osom

ycin

Dermaseptin 1

HipposinBuforinll

LAP

Drosocin

Pyrrhocoricin

Apidaecin

Indolicidin

Cath

elici

din

LL-3

7

Rana

tuer

in

Pexi

gana

n

Mag

aini

n 2

Cerco

pins

β-de

fensin

Bacte

necin

1Tha

natin

Tachyple

sinAndro

ctonin

Protegrin 1

α-definsin

θ-defensin

Histatin 5

Temporin

BMAP-28 (cathelicidin-5)

For the Antimicrobial Peptide Database see http://aps.unmc.edu/AP/main.php

http://aps.unmc.edu/AP/main.phphttp://aps.unmc.edu/AP/main.phphttp://aps.unmc.edu/AP/main.php


Review

various secondary metabolites. Genome plasticity in acti-nomycetes allows for stable maintenance of numerous biosynthetic gene clusters, including those encoding non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs).51 Members of the Gram-negative genus Xenorhabdus also possess a high number of NRPS and PKS genes in their genomes, representing a potential source for the discovery of new bioactive compounds. However, analysis of Xenorhabdus species has been limited by their distinctive lifecycle and symbiotic relationship with soil-dwelling nematodes.52 A new class of modified peptide antibiotics, the odilorhabdins, produced by enzymes of the NRPS gene cluster of Xenorhabdus nematophila was reported. Odilorhabdins show pro mising broad-spectrum antibacterial activity that relies on unconventional binding to a ribosomal site, which has not yet been used to develop therapeutics.53

A tripartite relationship exists to maintain homoeostasis between the host, microbiota, and other external colonisers through fine-tuned communication. The epithelial cells, phagocytic cells, and microbiota of the human gut, oral cavity, vagina, skin, and airways secrete AMPs that participate in host–microbiota crosstalk to promote a microbial and ecological balance. Their stability, potent antibacterial activity, and low toxicity to the host make these naturally produced AMPs potential drug candidates.54 In-silico mining of the human micro-biome using metagenomic libraries from the Human Microbiome Project and application of profile hidden Markov models have led to the detection of bacteriocin gene clusters in various niches in the human body.55 Bacteriocin gene clusters encoding ribosomally synthe-sised and post-translationally modified peptides are abundant in the human microbiome; for example, the thiopeptide lactocillin is produced by the vaginal commensal Lactobacillus gasseri and targets Gram-positive pathogens including S aureus and Gardnerella vaginalis.56 The non-ribosomally synthesised cyclic pep-tide lug dunin, produced by the nasal commensal Staphylococcus lugdunensis, selectively kills S aureus in the naso pharynx.57 A crucial question that remains un-answered is why most AMPs produced by Gram-positive commensals are selectively active towards Gram-positive pathogens. The answer is probably related to the term precision therapeutics, which refers to tailor-made molecules targeting specific pathogenic micro organisms or their virulence pathways, or both, without disturbing the surrounding microbiota. Pharma coge nomics will drive the future of precision medicine by investigating clinical therapeutic options targeting an individual’s genetic and epigenetic makeup.58

Broadening methodological drug designDespite the enormous diversity in amino acid sequences and cellular targets of known AMPs, they are unified by enhanced affinity for negatively charged prokaryotic membranes with strong electrical potential gradients as

prerequisites for cellular entry or direct disruption of the bacterial cell membrane.59 One of the best understood peptide antibiotics, gramicidin or gramicidin D, is a mix of the non-ribosomal gramicidins A, B, and C and is atypically neutral and highly hydrophobic. Its sequence features alternating L and D amino acids that form a head-to-head β-helical dimer with a large lumen that allows ion movement across the membrane, leading to disruption.60 Although additional contributions to killing have been identified, such as hydroxyl radical production, these events appear to be downstream consequences of membrane per meabilisation.61 Conversely, gramicidin S is more typical, being a cyclic, cationic, amphipathic, and decameric peptide.62 The mechanisms of typical animal-derived AMPs—such as the helical magainin,63 the β-hairpin protegrin,64 or the disordered indolicidin65—and their interaction with membranes and nature of the channel state are unclear.66 Extensive experimental efforts have been geared towards investigating these questions, including monitoring fluorescent dye leakage from vesicles;67 fluorescence microscopy imaging of giant unilamellar vesicles68 and of live cells,69 calorimetry,70 oriented circular dichroism spectroscopy,71 x-ray diffraction,72 neutron scattering,73 electrophysiology,74 solution nuclear magnetic resonance in detergent micelles,75 solid-state nuclear magnetic resonance in native environments,76 and atomic force and electron microscopy.77,78 However, a high-resolution structure of an AMP-stabilised pore is still not available. The interaction of AMPs with membranes has also been extensively studied by molecular dynamics simulations.79 Although membrane permeabilisation by AMPs is widely accepted as one of the possible mechanisms, clustering of ionic lipids,80 displacement of peripheral membrane proteins,81 interference with cell wall biogenesis,82 and targeting intracellular components, have also been proposed.83

The mentioned constraints identify paths towards the discovery and design of AMPs with relevant properties (figure 3). However, the search for novel AMPs is complicated by two other factors. First, peptides of 50 residues or fewer have more than 10⁶⁵ unique sequences. Edisonian trial-and-error experimentation can sample only a small proportion of the available sequence space. Second, although some of the physi-cochemical properties of naturally occurring AMPs are known, there might be other common features that are harder to recognise which are crucial for the biological function of AMPs.85 These two challenges have led to the development of in-silico models for virtual high-throughput screening of AMPs based on the spacial orientation of peptide sequences. Large databases, high-performance com puting, and machine learning algorithms enabled the emergence of cheminformatics and quantitative structure-activity relation models of AMP activity in the mid-2000s.85,86 Machine learning models have been used for these developments,


Review

including support vector machines, hidden Markov models, random forests, and artificial neural net-works.85,87–89 Some models are ideal for high-accuracy, high-throughput screening and have been successful in the discovery of AMPs with higher antimicrobial potency than those AMPs that have entered clinical trials.87,90 Other models are better suited to reveal the universal features that determine the biological activity of AMPs and have advanced the mechanistic understanding and unveiled the membrane activity of diverse families of AMPs.89,91 More sophisticated modelling approaches could combine quantitative structure-activity relation models with network approaches using a robust screening dataset. The use of cheminformatics in the design of AMPs with multiplexed functionality such as immunomodulation, signalling, or toxicity in addition to membrane activity has also emerged as a powerful strategy to design novel antimicrobials.13

Advances in the development of computational tools have facilitated the discovery of novel peptide-based antibiotics.92,93 Data mining approaches have led to the discovery of pepsin A, a novel AMP from previously unexplored sources, on the basis of experimentally validated anti microbial scoring functions.94 Structure–function guided investigation of the toxic peptide polybia-CP derived from wasp venom has led to the identification

of crucial activity determinants such as physicochemical features, which allowed precise manipulation of the peptide’s antimicrobial and cytotoxic activities.95 Progress in recombinant DNA technology is being made towards large-scale production of AMPs at affordable prices, which could lead to improved AMP biomanufacturing platforms. Gaglione and colleagues96 implemented the novel Notomista-Arciello broth that yields increased cell biomass and recombinant expression to obtain large-scale production of host defence peptides combined with the carrier protein onconase (protein P-30). The pro duction cost of recombinant chimeric constructs ONC-r(P)GKY20, ONC-r(P)ApoBL, and ONC-r(P)ApoBS decreased from €253 to €42 per mg when the scale increased from 100 mg to 1000 mg per batch, showing that cost reduction is associated with labour task alleviation. The genetically engineered yeast Pichia pastoris has been successfully used for affordable, high, and heterologous production of recombinant apidaecin IA in bioreactors that yielded approximately 1 g of apidaecin at a cost of only US$1.97

The use of mixture-based synthetic combinatorial libraries permits systematic screening of millions of peptides for antimicrobial efficacy in microdilution assays (table 1). Although millions of compounds can be readily screened using combinatorial approaches, the actual

Figure 3: Mechanisms for interaction of AMPs with bacterial cell membranesVarious events occurring at the bacterial cell membrane following initial interaction with AMPs. Examples of AMPs for each mechanism of action are shown.84 AMP=antimicrobial peptide. PGLa=peptide glycine-leucine-amide.

H

O

Electroporation (eg, NK-lysin) Non-lytic membrane depolarisation(eg, bovine lactoferricin, daptomycin)

Toroidal pore (eg, magainin 2,melittin, protegrin I)

Carpet model (eg, cercopin, aurein)

Oxidised lipid targeting(eg, temporin L, indolicidin)

Barrel stave (eg, alamethicin)

Disordered toroidal pore(eg, magainin analog, melittin)

Non-bilayer intermediate (eg, gramicidin S)Membrane thinning or thickening (eg, PGLa,cathelicidin LL-37)


Review

screening of libraries requires only 300–600 samples. Furthermore, the generation of diverse chemical libraries using an approach combining combinatorial libraries with the post-synthetic chemical modification of existing peptide libraries have enabled the creation of single screening assays, the generation of peptidomi-metics libraries, and low mole cular weight organic compound librar ies.98–100 Screening of mixture-based combinatorial libra ries has resulted in the identifi cation of bis-cyclic guani dines with broad anti microbial activity against all antibiotic resistant ESKAPE pathogens (Enterococcus faecium, S aureus, Klebsiella pneu moniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) at concentrations of less than 2 μM.101 In addition, these compounds showed bactericidal and antibiofilm properties with reduced potential for the development of resistance and showed almost no toxicity to human lung cells and erythrocytes.102–104 Identi fying new antibacterial agents against ESKAPE patho gens using mixture-based combinatorial approaches includes creating lipopeptides with improved anti microbial potency to disrupt bacterial biofilm formation, potentiating anti-biotics, reversing resist ance of patho gens to antibiotics, and developing efflux-pump inhi bitors to block active

extrusion of antibiotics.94,95 More over, screening of mixture-based libraries in target-based assays has also resulted in the identification of antimicrobial compounds, including peptides and small molecule libraries that inhibit different steps of site-specific recombination, particularly the accu-mulation of Holliday junctions (N-methyl aminocyclic thiourea inhibitor peptides)105 and type IB topoisomerases (peptides, polyamines).106

The pharmacological profiles of AMPs still remain largely unknown. Using pharmacodynamics based on the Hill function, strong synergistic effects of combining two or three peptides on bacterial killing have been reported.107 A pharmacodynamics approach to evaluate combinations can lead to improved evaluation of AMPs in practice, besides providing insight into the evolution of AMPs interactions with the immune system. The emergence of resistance can be tracked at specific con centrations of AMPs, and their effectiveness as marketed drugs can be determined to reduce production cost and toxicity risk.107 Databases provide easy, rapid, and cost-effective ways to predict and discover novel peptides with potent activity on the basis of reliable sources, patents, and literature.86 Although no specific algorithms exist to predict AMP toxicity, Hemolytik is a manually curated database with information on the haemolytic activity of AMPs gathered from scientific literature, The Anti microbial Peptide Database,108 Collection of Antimicrobial Peptides Database,109 Dragon Antimicrobial Peptide Database,110 and UniProtKB.111 Thus, the spectrum of prediction is limited to existing knowledge.112

Peptidomics traces the peptides present in biofluids of an organism at the time of analysis and elucidates the structural and functional features to map their bioactivities on the basis of naturally-derived protein fragments. Proteasix is an open-source peptide tool addressing AMP-generating proteases and their activity, in combination with peptidomic databases.113 The isobaric tag for relative and absolute quantitation (iTRAQ) offers a sensitive quantitative method to detect post-AMP treatment changes in protein expression and identify molecular pathways of bacterial growth inhibition and killing.114 For example, gene expression profiling of the periodontopathogen Porphyromonas gingivalis following AMP treatment with Nal-P-113 suggested that new molecular pathways are involved in the inhibition of oral biofilm formation associated with AMP administration.115 Mass spectrometry-based proteomics complemented by collision-induced dissociation, electron-capture disso-ciation, and electron-transfer dissociation fragmentation methods provide a top-down approach in generating large-scale datasets that could surpass the technical limitations and laborious methodologies which hinder the discovery of AMPs from complex biological materials.116,117

Recent developments in deep learning methods and convolutional neural networks have allowed for the prediction of compound toxicity from simple 2D drawings of chemicals.118 Realistic graphical representations of

Creation Summary

TANGO 2004 TANGO software computes in-vitro aggregation of proteins

GROMACS 2005 Versatile package for molecular dynamics

AGGRESCAN 2007 AGGRESCAN software computes in-vivo aggregation of proteins

CHARMMing 2008 Molecular simulation program

HeliQuest 2008 A web server to screen sequences with specific α-helical properties

ExPASy 2012 Resource portal providing access to scientific databases and software

ProtParam 2012 Allows the computation of physical and chemical variables for a given protein

ProPAS 2012 Stand-alone software to analyse protein properties

YADAMP 2012 Database of antimicrobial peptides based on an extensive literature search

LAMP2 2013 Database of antimicrobial peptides that provides a useful resource and tool for studies

CPPpred 2013 A web server for the prediction of cell-penetrating peptides

ToxinPred 2013 A web server for designing and predicting toxic peptides

Hemolytik 2014 Manually curated database of experimentally validated haemolytic and non-haemolytic peptides

3D-HM 2014 Tool to characterise the surface polarity of amphiphilic peptides

APD3 2016 The antimicrobial peptide database

CAMPR3 2016 Collection of antimicrobial peptides

DBAASP v.2 2016 Database of antimicrobial activity and structure of peptides

Helixator 2016 A web tool that facilitates the identification of amphipathic protein transmembrane segments

SATPdb 2016 Database of structurally annotated therapeutic peptides

pepATTRACT 2017 Computational docking tool to study peptide–protein interactions

BAGEL4 2018 A genome mining tool for putative bacteriocin gene-cluster detection

DRAMP 2·0 2019 Database containing general, patent, and clinical antimicrobial peptides

Links to each website can be found in the appendix.

Table 1: Databases and bioinformatics resources for antimicrobial research

See Online for appendix


Review

peptide surfaces can capture bacterial cell-wall disruption by AMPs through surface-mediated electrostatic and hydrophobic interactions. The improved predictive ability of cheminformatics allows optimisation of AMPs using surface recognition, quantitative structure-activity relation modelling. To produce realistic graphical representations of the 3D surface, self-organising map transformation yields a 3D to 2D mapping framework for deep neural network training.119 Surface characteristics that control peptide effectiveness can be learned using 2D convolutional nets. The availability of online databases, experimental modelling techniques for structure-activity relationships, and theoretical methodologies such as computational algorithms, software, and prediction programs have advanced design of AMPs.120–122 Notably, these techniques have revealed that short AMPs exhibit a better pharmacological profile, higher metabolic stability, low toxicity, and a lower production cost compared with long AMPs.123

The relation between the peptide sequence and activity is complex, as revealed by the use of various structural and inductive descriptors in high-throughput quantitative structure-activity relation studies, but some generali-sations can be made.88 Alterations in peptide cationicity and hydrophobicity affect the balance between bacterial killing and toxicity. Increased net charge enhances anti-microbial activity through electrostatic force-associated interactions with bacterial membranes, but excessive charge can promote undesirable haemolysis.124 Amphi-pathicity is strongly related to the net charge and hydrophobicity of AMPs and affects their antimicrobial activity, target selectivity, and toxicity to eukaryotic cells. Reduced helicity, achieved by substitutions of L and D amino-acid enantiomers is important in lowering the haemolytic activity of AMPs.125 Acetylation and amidation of the N and C termini can also protect AMPs from degradation by proteases, but N-acetylation reduces overall net charge, whereas amidation favourably increases the positive charge.126,127 PEGylation is another chemical method to enhance the half-life and bioavailability of AMPs.128 Cyclisation using click chemistry stabilises large peptide structures and enhances metabolic stability, although the effect on antimicrobial activity and host toxicity is still largely unknown.129 Finally, the positioning rather than the number of cationic residues affects the therapeutic index of AMPs. This variable is indicative of AMP selectivity towards the bacterial membrane over the zwitterionic eukaryotic cell membrane, thus affecting their therapeutic abilities.130

Nanoscale delivery of AMPsOptimisation strategies in the design of modern AMP-polymer surfaces aim to increase specificity, enhance accumulation and biocompatibility, stability, prolong site-specific retention, and reduce toxicity. Nanostructures including micelles, vesicles, nanoparticles, and nano-sheets that are loaded with AMPs conjugated to polymers

have been proposed to adapt stimuli from different microenvironments (pH, redox, enzyme, temperature). Simplicity and cost-effectiveness of the optimisation process (coating or encapsulation) and the ability to overcome shortcomings of components when applied alone enhance the industrial practicality and commercial viability of the end-product, offering added value in smart biomedical applications.131 Chimeric peptide analogues have shown antimicrobial and anti biofilm properties against Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli when conjugated with titanium alloys in orthopaedic and oral implant-related infections by creating contact-killing surfaces via modulation of bacterial gene expression and suppression of colo-nisation.132,133 AMP-releasing coatings (hydrogels, poly-mers, microporous calcium phosphate coatings) have been tested in endotracheal tubes, sutures, and catheters showing the ability to prevent infection by a single species or polymicrobial biofilms and to reduce endotoxin release.134,135

Chitosan-based formulations with added peptido-mimetics in the form of a toothpaste have shown potential in daily routine of oral and dental care.136 The MSI-78 synthetic analogue, from the family of magainin 2 AMPs, immobilised onto a maleimide surface using silica coating has been studied for bacterial sensing and killing properties. On contact, the vertically standing immobilised MSI-78 killed E coli BL21 (DE3) competent cells through charge interaction and not by the usual penetration of the bacterial membrane by AMPs in free solution (barrel-stave or toroidal pore model).137 In a mouse model, HHC-36 AMP encapsulation into a dual layer of anhydrous polycaprolactone polymer-based coating showed antibacterial and wound-healing properties, with good biocompatibility and controlled release of AMPs. This finding could be a potential prevention measure against device-associated infections in daily clinical practice.138 Vancomycin-loaded poly(ε-caprolactone) scaffolds were formulated using supercritical CO2 foaming normally used for bone regeneration. The scaffolds showed consistent vancomycin release for more than 2 weeks, with anti-microbial and antibiofilm efficacy against S aureus, and they also enhanced osteodifferentiation and cellular pro-liferation, which could potentially reduce the risk of implant-associated infections.139

Nanotubes, quantum dots, graphene, and metal nanoparticles that form porous structures loaded with AMPs have been proposed to enhance drug delivery. The chimeric peptides KG18 and VR18, conjugated with tungsten disulphide quantum dots, showed antibacterial and antibiofilm activity against P aeruginosa, with a favourable toxicity profile.140 Structurally nano-engineered AMP polymers did not develop resistance and showed in vitro and in vivo efficacy against E coli, P aeruginosa, K pneumoniae, and A baumannii.141 Coating nanoparticles that either encapsulate or bind to AMPs not only increased antibacterial efficacy but also enhanced bioavailability


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and escaped proteolytic activity or bacterial efflux.142,143 Polylactide-co-glycolide nanoparticles encapsulating the esculentin-1a-derived AMPs such as Esc(1–21) and Esc(1–21)-1c have been proposed as a successful lung-delivery system.144 Antifouling surfaces include functionali sation of hydrophilic polymer coatings poly(ethylene glycol) or oligo(ethylene glycol) coatings, or polymer brush coatings with AMPs that display anti-adhesion and antibiofilm effects against a broad spectrum of bacterial species. This finding suggests a potential alternative hybrid catheter material for catheter-associated urinary tract infections, catheter-related bloodstream infections, pre vention, and wound healing with increased therapeutic potential, reduced toxicity, and prevention of proteolytic degradation.145–148 Nanocarriers as an intelligent delivery system have only been evaluated in vitro and in animal models.149,150 Intense development is ongoing to find new ways to scale up and do quality controls of nanocarriers involving nanoparticles or PEGylated micelles, so that these technologies can be successfully introduced into clinical practice. Finally, recombinant DNA technology was successfully used in engineering a biomaterial based on spider silk, human neutrophil defensins 2 (HNP-2 and HNP-4), and hepcidin showing antimicrobial efficacy against S aureus and E coli.151 Incorporation of AMPs into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes showed strong antimicrobial activity against S aureus and K pneumoniae (4–6 log reduction) and reduced cytotoxicity to healthy human dermal fibroblasts.152

Synergism with conventional antibiotics: can AMPs overcome antimicrobial resistance?Studies over the past decade have investigated synergism between AMPs and conventional antibiotics.153 Previous research is not sufficiently statistically robust and does not fully support the hypothesis that intracellularly acting conventional antibiotics synergise with the membra-nolytic activity of AMPs.154 Underlying molecular mecha-nisms and the cross-cytotoxic effects driven from the interaction of AMPs with antibiotics remain unexplored. For example, the combination of AMPs with an established membra nolytic effect (porcine protegrin 1, human β-defensins, LL-37) with intracellularly targeting antibiotics (genta micin, ofloxacin, rifampicin) against MRSA, Micrococcus luteus, A baumannii, K pneumoniae, P aeruginosa, and E coli did not show specific patterns regarding cytotoxic and haemolytic effects.155 The combination of anthelmintic salicylanilides (oxyclo-zanide, rafoxanide, closantel) with colistin has been proposed as an effective killing strategy against multidrug-resistant Gram-negative clinical isolates (A baumannii, K pneumoniae, P aeruginosa, E coli) that would overcome the disadvantages of poor membrane permeability and efflux, as shown by the use of salicylanilides as monotherapy.156 The ability of

five different polycationic peptides (buforin 2, cecro-pin P1, indolicidin, magainin 2, and ranalexin) to enhance antibiotic activity in vitro was also explored.157 Giacometti and colleagues157 showed that combi nations of renalexin potentiate the activity of polymyxin E, doxycycline, and clarithromycin. Magainin 2 also elicited synergistic potentiation (4–8 times) of ceftriaxone, amoxicillin–clavulanate, ceftazidime, meropenem, and piperacillin. However, buforin 2, cecropin P1, and indolicidin did not potentiate the activity of antibiotics.

Otvos and colleagues158 found that AMPs act additively and synergistically when combined with carbapenems (imipenem) in vitro. In addition, when the proline-rich antibacterial peptide A3-APO was combined with imipenem, it increased survival in vivo in a murine melioidosis model. When imipenem and A3-APO were combined, 80% of mice survived for more than 36 h after infection, compared with either monotherapy that resulted in only 40% survival. A recent study159 showed synergy of antibiofilm peptides and various antibiotics when tested against each of the ESKAPE pathogens in a high-density infection animal model. The promising potential for peptides to synergise with antibiotics has been clinically investigated in a randomised controlled trial. Paul and colleagues160 investigated the use of cationic lipopeptide colistin in combination with meropenem to evaluate whether the therapy improved the clinical outcome against carbapenem-resistant A baumanii infections. Although the combination of colistin and meropenem is synergistic in vitro, there was no significant improvement in the clinical outcome when compared with colistin monotherapy. Nevertheless, other combinations with polymyxins have been shown to be synergistic in animal models.161 The synergistic activity of daptomycin combined with β-lactams showed efficacy of 90% in combination versus 57% in monotherapy (p=0·061) in patients with staphylococcal bacteraemia associated with infective endocarditis or bone and joint infection.162 Coadminis tration of daptomycin with linezolid showed therapeutic effects in intravenous drug users with persistent MRSA right-sided infective endocarditis. Complete resolution and negative blood cultures were obtained after 13 days of the combined therapeutic regimen administration.163 The benefits of successful AMP coadministration included reduction in hospitalisation length, less absence from work because of illness, and reduced treatment costs resulting in a more financially sustainable health-care system.

Besides the ability to synergise with antibiotics, the antimicrobial potential of AMPs can be enhanced by exploring synergies among different AMPs, which would reduce the risk of antimicrobial resistance development.107 The combination of multiple AMPs (cecropin A, LL 19-27, melittin, pexiganan, indolicidin, and apidaecin) showed a pharmac odynamically compatible synergistic effect in killing E coli in vitro. Triple AMP combinations (pexiganan and LL 19-27 plus melittin; melittin and indolicidin plus LL 19-27; melittin and cecropin A plus


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LL 19-27; melittin and apidaecin plus LL 19-27), showed an increase of 85% synergistic killing effect compared with a 67% increase with double-AMP combinations

(LL 19-27 and cecropin A; pexiganan and indolicidin; melittin and LL 19-27; pexiganan and melittin).107 Host AMPs have also been found to synergise with metabolites

Clinical application Target and mechanism of action

Route of administration

Developmental stage (decision)

Company

Human lactoferrin-derived peptide hLF1–11

LPS-mediated diseases and fungal infections

DNA-binding Intravenous Phase 1 (completed) AM-Pharma

Histatin-1 and 3, P-113 (histatin derivatives)

Chronic Pseudomonas aeruginosa infections, gingivitis, and periodontal diseases

Reactive oxygen species generation

Topical (gel mouthwash)

Phase 1, Phase 2–3 Demgen

LTX-109 (lytixar) Uncomplicated Gram-positive skin infections, methicillin-resistant Staphylococcus aureus, and methicillin-sensitive S aureus nasal carriage

Membrane permeabilisation

Topical Phase 2 Lytix Biopharma

Opebacan (rBPI21, neuprex)

Meningococcal, wound, and burn infections


Intravenous Phase 2 (completed) Xoma

Opebacan (rBPI21, neuprex)

Post-traumatic infections Membrane permeabilisation

Intravenous Phase 2 (failed) Xoma

Omiganan (MBI-226, MX-594AN)

Catheter-associated infection Membrane permeabilisation

Topical Phase 3 (completed) Migenix

Omiganan (MBI-226, MX-594AN)

Severe acne, rosacea, genital warts, and vulval epithelial neoplasia

Anti-inflammatory Topical Phase 2b (completed) Migenix, Biofrontera

EA-230 Sepsis, endotoxemia Immunomodulation Intravenous Phase 2 (recruiting) Exponential Biotherapies

IMX942 (dusquetide)

Oral mucositis Immunomodulation Topical Phase 3 (recruiting) Inimex Pharmaceuticals

PMX-30063 (brilacidin)

Acute bacterial skin infections Membrane permeabilisation

Intravenous or topical

Phase 2 Innovation Pharmaceuticals

OP-145 Chronic otitis media Bacterial toxins neutralisation

Topical (ear drops) Phase 2 (completed) OctoPlus

XF-73 Surgical site staphylococcal infection, nasal carriage


Topical Phase 2 (recruiting) Destiny Pharma

XOMA-629 Impetigo Membrane permeabilisation

Topical (gel) Phase 2a Xoma

DPK 060 Eczematous lesions infection NA Topical Phase 2 (completed) DermaGen AB

Murepavadin (POL7080)

P aeruginosa-associated lower respiratory infection and ventilator-associated pneumonia

Outer membrane lipopolysaccharide transport protein D

Intravenous Phase 3 (recruiting) Polyphor

Surotomycin Clostridium difficile-associated diarrhea

Membrane depolarisation

Oral Phase 3 (completed) Cubist Pharmaceuticals, Merck

Iseganan (IB-367)

Oral mucositis, ventilator-associated pneumonia


Mouthwash, aerosols

Phase 3 (failed) IntrabioticsPharmaceuticals

XMP 629 Acne NA Topical Phase 3 (failed) Xoma

Talactoferrin (TLF, rhLF)

Sepsis NA Oral Phase 3 (suspended) Agennix

Pexiganan (locilex)

Diabetic foot ulcer infections Membrane permeabilisation, defensin stimulation


Phase 3 (failed) Genaera

Pexiganan acetate (Locilex, MSI-78, or Pexiganan acetate)

Impetigo, diabetic ulcer infections Membrane permeabilisation, defensin stimulation


Phase 3 (failed) Genaera

p2TA (AB103) Necrotising soft tissue infection Immunomodulation Intravenous Phase 3 Atox Bio

Suspended means the study was stopped early but might start again. Completed means the study ended as planned. This list is not extensive. A complete list of clinical data on AMPs can be found on the Data Repository of Antimicrobial Peptides. AMPs=antimicrobial peptides.

Table 2: AMPs in clinical trials as anti-infective therapies

For the Data Repository of Antimicrobial Peptides see http://dramp.cpu-bioinfor.org/browse/ClinicalTrialsData.php

http://dramp.cpu-bioinfor.org/browse/ClinicalTrialsData.phphttp://dramp.cpu-bioinfor.org/browse/ClinicalTrialsData.phphttp://dramp.cpu-bioinfor.org/browse/ClinicalTrialsData.php


Review

produced by symbiotic bacteria to protect the host against pathogens.164

What is holding up the clinical pipeline?Despite the alarming increase in antimicrobial resistance, most pharmaceutical companies have abandoned the development of new antibiotics and have instead focused on the development of more profitable drugs to treat non-communicable diseases. However, considering that bacteria kill in the short term, giving priority to treating non-communicable diseases in the long term is questionable if infection-driven mortality rates cannot be halted in the short term. Most AMPs currently in clinical trials are analogues or modified derivatives of natural peptides. AMPs were introduced in clinical practice in 1939 when gramicidin and tyrothricin were shown to be active against some Gram-negative bacteria, including Neisseria species. First used clinically in 1959, towards the end of the 20th century, the polymyxin antibiotic class with its annotated dose-dependent nephrotoxicity had once again started being used to combat superbugs. The well known colistin now remains the last viable therapeutic option for Gram-negative bacteria.165 However, in late 2015, the first plasmid-mediated polymyxin-resistance gene (MCR-1) was reported, indicating that this last-line antibiotic is losing its effectiveness, and the world was confronted with a revelation of the antibiotic resistance crisis.166

Attempts to identify and design clinically relevant AMPs have led to more than 2800 molecules being identified, of which only a few have proceeded to preclinical studies, clinical trials, have obtained US Food and Drug Administration approval, or have been launched on the

market (table 2). The cyclic decameric peptide gramicidin S and the polymyxin B lipopeptide used in combination with bacitracin or with neomycin and bacitracin are highly used over-the-counter drugs, whereas nisin is mostly used in Europe as an antibacterial food additive. Pexiganan (MSI-78), iseganan (IB-367), omi ganan (MBI-226), other synthetic analogues of indolicidin, and neuprex (rBPI21) did not show the expected effecitveness in phase 3 clinical trials. The trials were not successful mainly because of flawed trial design, inability to meet targets for clinical effectiveness, and the absence of improved activity over conventional antibiotics, or, in the case of iseganan, increased mortality compared with placebo.167 Although AMPs disrupting the membrane show broad-spectrum antibacterial activity in vitro, they have also resulted in systemic and local toxicity hampering the successful transition from bench to bedside (table 2). Non-pharma-cological causes of failure include instability of formulated peptides, confounding biological activities of peptides (eg, pro-inflammatory and anti-inflammatory effects), and high manufacturing costs. Accordingly, the major goal for biotechnological and pharmaceutical companies is to develop AMPs that are highly selective against pathogenic bacteria, exert no harm to the host cells at therapeutic doses, possess a good pharmacological profile, evade resistance development by the pathogen, and are cost-effective in terms of pharma ceutical develop ment. Choosing the appropriate synthetic technology on the basis of peptide sequences and product volumes (solid-phase, liquid-phase, or hybrid) is the key to scale down the manufacturing cost.168 In the past 5 years, synthetic lipopeptides murepavadin (also known as POL7080; NCT03409679) and surotomycin (NCT01597505, NCT01598311) have success fully entered phase 3 clinical trials. Murepavadin represents a novel class of antibiotics that target the lipopolysaccharide transport protein D in the outer membrane of Gram-negative bacteria. Murepavadin is specifically potent against P aeruginosa-associated noso comial pneu monia.169 Surotomycin is a calcium-depen dent antibiotic with Gram-positive and Gram-negative bacterial membrane-depolarising properties and it is being evaluated as an emerging treatment for C difficile-associated dia rrhoea.170 Optimisation of the macrocyclic lipopeptide arylomycin resulted in the promising derivative G0775. G0775, developed by Genentech (South San Francisco, CA, USA), inhibits bacterial type 1 signal peptidase with increased target affinity and successfully permeabilises the bacterial outer membrane by avoiding AcrAB-TolC multi drug efflux pumps. Once they receive regulatory approval, these clinical molecules would provide validation to further investigate peptide-based molecules to treat antibiotic-resistant bacterial infections.

ConclusionSince the discovery of nisin in the late 1930s, many peptides have been identified as antimicrobials with different efficacy profiles. Chemical versatility is both a benefit and a drawback for pharmaceutical and clinical

Search strategy and selection criteria

References for this Review were identified through non-confidential information on projects and inventions provided by its members, comprehensive scientific literature, and database searches to identify research articles, clinical trials, publications, and companies. Preclinical and clinical projects were identified using searches of PubMed for articles published from Aug 1, 1987, to July 28, 2019, and the ClinicalTrials.gov database up to Aug 30, 2019, by use of key terms such as “antimicrobial peptides”, “bacterial killing”, “biofilm”, “resistance”, “patent”, “drug delivery”, “design”, and “machine learning models”. A refinement of current bibliography in the World Intellectual Property Organization (WIPO) using the key terms “antimicrobial peptide” AND “human” AND “antibacterial” ANDNOT “virus” ANDNOT “fungi” ANDNOT “plant” ANDNOT “animal”, resulted in 51 published inventions regarding antimicrobial peptides. Relevant articles were identified through searches in the authors’ personal profiles in Google Scholar. Relevant articles identified by this search were included. Articles and inventions published only in English were included.


Review

development, as there are many plausible drug targets. AMP use requires the alignment of microbiology, medicinal chemistry, and preclinical development strategies, but there is little information available, especially in the preclinical area (pharma cokinetics, formulation, and Absorption, Distribution, Metabolism, Excretion and Toxicity [ADME-Tox] studies), to guide development. Synthetic peptides stemming from the combined knowledge of peptide sequences and physi-cochemical properties have numerous unknowns, particularly with respect to systemic toxicity. A rationalised synthetic strategy can overcome some of the limitations of natural sequences in eliminating multidrug-resistant and pandrug-resistant bacterial isolates in vitro.

AMP commercialisation favours topical over systemic administration, further posing a barrier for the launch of peptides that can be used as drugs in the market. The methodologies of experimental and theoretical pharma-cology should align with translational research to provide targeted information for rational synthesis, structure-activity relationships, resistance patterns, and relevant toxicity, and to validate bioavailability assays. The use of genetic algorithms to computationally evolve natural AMPs into synthetic derivatives is a concept that is moving to mouse models, showing that computational approaches could be used to generate novel antibiotics. To date, no validated reports exist regarding the timeframe required for a bacterial population to become resistant to AMPs. Laboratory and clinical studies have provided some information, enabling geno type analysis to bridge the apparent gap from bench to bedside. The microbiome concept is a classic example that has dominated the literature regarding the role of AMPs in health and disease. Filtering this information to adhere to novel discovery concepts will require matching to robust epidemiological, environmental, and drug develop ment data. Synergistic approaches that include AMPs and conventional anti-biotics will expedite clinical development, creating the need for clinical trials.ContributorsGPT defined the focus of this Review. MM, MP, and GPT prepared the first draft. All authors contributed to scientific literature searching and interpretation of findings, according to their areas of expertise. ALS, LL, MF, AI, MAG, YA, SB, ALF, AC, MNS, CP, CdlF-N, TL, TD, RAH, and REWH edited the draft before wider input. MM and GPT finalised and submitted the manuscript.

Declaration of interestsWe declare no competing interests. REWH has more than 50 patents covering specific antimicrobial peptides, assigned to his employer University of British Columbia that has licensed these to several companies, which has the potential for Hancock to earn royalty payments. One of these companies is ABT Innovations, in which REWH and CdlF-N have ownership interests. No AMPs reviewed were produced by ABT Innovations or patents owned by REWH and CdlF-N. GPT has moved from GAMA Therapeutics (Mansfield, MA, USA) to Reading Hospital, Tower Health (West Reading, PA, USA) on June 22, 2020.

AcknowledgmentsALS is supported by a Marie Curie Global Fellowship (EU project 843116). GPT mentors ALS in the Fellowship. CdlF-N holds a presidential professorship at the University of Pennsylvania, a Langer Prize by the

AIChE Foundation, and acknowledges funding from the Institute for Diabetes, Obesity, and Metabolism, and the Penn Mental Health AIDS Research Center of the University of Pennsylvania. TL was supported by the National Institutes of Health (RO1 GM117146). REWH peptide research is supported by a Canadian Institutes for Health Research grant (FDN-154287), by a Canada Research Chair, and a UBC (University of British Columbia) Killam Professorship.

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