antibiotics Review Synergies with and Resistance to Membrane-Active Peptides Adam Kmeck, Robert J. Tancer , Cristina R. Ventura and Gregory R. Wiedman * Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ 07079, USA; [email protected] (A.K.); [email protected] (R.J.T.); [email protected] (C.R.V.) * Correspondence: [email protected] Received: 24 August 2020; Accepted: 17 September 2020; Published: 19 September 2020 Abstract: Membrane-active peptides (MAPs) have long been thought of as the key to defeating antimicrobial-resistant microorganisms. Such peptides, however, may not be sufficient alone. In this review, we seek to highlight some of the common pathways for resistance, as well as some avenues for potential synergy. This discussion takes place considering resistance, and/or synergy in the extracellular space, at the membrane, and during interaction, and/or removal. Overall, this review shows that researchers require improved definitions of resistance and a more thorough understanding of MAP-resistance mechanisms. The solution to combating resistance may ultimately come from an understanding of how to harness the power of synergistic drug combinations. Keywords: membrane-active peptides; antimicrobial-resistance; drug synergy 1. Introduction Membrane-active peptides (MAPs) are peptides ranging from about 4–40 amino acids in length that can interact with the cell membrane through permeabilization or other antimicrobial mechanisms [1,2]. They are often comprised of amino acid residues that are positively charged at pH 7. They can be grouped into four main structural categories: Linear α-helices, extended structures (usually abundant in Glycine, Arginine, Tryptophan, or Proline residues), β-sheets (often stabilized by disulfide linkages), and loops that contain both α and β moieties [3]. Table1 shows examples of these peptides that are discussed in this review. In the past 10 years publication of materials concerning MAPs has exponentially increased [2]. This increase is partly due to MAPs potential ability to combat antimicrobial-resistance [4]. MAPs are found in numerous organisms ranging from humans (α-defensin) to insects (cecropin A) [2]. Extensive studies in this field have shown that there are many varied mechanisms of action. Proposed mechanisms include: The formation of toroidal and barrel stave pores, as well as non-pore forming mechanisms, such as carpet, detergent, inverted micelle, and membrane thinning models [1,2,5]. All these mechanisms contribute to the destabilization of lipid membranes in one manner or another. Table 1. Sequences of membrane-active peptides (MAPs). Peptide Sequence Melittin GIGAVLKVLTTGLPALISWIKRKRQQ Pixiganan GIGKFLKKAKKFGKAFVKILKK CAP18 GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES K5L7 KLLLKLKLKLLK Antibiotics 2020, 9, 620; doi:10.3390/antibiotics9090620 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 15 AntibioticsDisruption2020, 9,of 620 the cell membrane of a microorganism can have many deleterious2 ofeffects. 15 Biophysical studies have long supported the hypothesis that MAPs cause depolarization of the cell membrane,Disruption which removes of the cell the membraneelectrochemical of a microorganism gradient needed can to have drive many molecular deleterious transport effects. across the membraneBiophysical [6,7] studies. An have additional long supported role may the be hypothesis played by that the MAPs ability cause of depolarizationMAPs to corral of theanionic molecules,cell membrane, specifically which charged removes lipids, the electrochemical leading again gradient to disruption needed toof drivetransport molecular and disruption transport of signalacross transduction the membrane [8–10] [6,7. ]Total. An additional disruption role of may the bemembrane played by theand ability escape of MAPsof large, to corralmacromolecules anionic has beenmolecules, seen specificallyand an important charged lipids,mechanism leading with again synthetic to disruption MAPs of transport [11]. Researchers and disruption in this of signalarea have transduction [8–10]. Total disruption of the membrane and escape of large, macromolecules has been repeated the claim that, no matter what the exact mechanism, the ability of MAPs to target the cell seen and an important mechanism with synthetic MAPs [11]. Researchers in this area have repeated membrane allows them to either partially or totally circumvent the development of resistance [12– the claim that, no matter what the exact mechanism, the ability of MAPs to target the cell membrane 14]. allowsIn this them review to either, we partially explore or the totally literature circumvent surrounding the development resistance of resistance to MAPs [12–, 14and]. In we this offer suggestionsreview, we for explore ways forward the literature to address surrounding those resistance possible toavenues MAPs, andof resistance we offer suggestions. for ways forwardIn thinking to address about those the possibleway MAPs avenues interact of resistance. with the cell membrane, we would define possible resistanceIn and thinking synergy about in thefour way stages MAPs (Figure interact 1). withThe thefirst cell stage membrane, would be we when would the define peptides possible are still outsideresistance of the andcell. synergy At this inpoint, four they stages are (Figure subject1). to The peptidases, first stage wouldwhich bemay when degrade the peptides them [15] are . The use stillof MAPs outside could of the cell.select At for this organisms point, they arethat subject have tomutated peptidases, extracellular which may peptidases, degrade them enhanced [15]. extracellularThe use of peptidase MAPs could synthesis select for pathways, organisms improved that have mutated transport extracellular of peptidases peptidases, to the enhanced extracellular space,extracellular or a combination. peptidase synthesisThe second pathways, stage improved is at the transport cell wall of o peptidasesr outer membrane to the extracellular of the space,cell. This or a combination. The second stage is at the cell wall or outer membrane of the cell. This structure often structure often contains charged molecules that can bind to MAPs and prevent them from reaching contains charged molecules that can bind to MAPs and prevent them from reaching the cell membrane. the cell membrane. The third stage is when the peptide interacts with the lipids in the cell membrane. The third stage is when the peptide interacts with the lipids in the cell membrane. The assumption in The assumptiondesigning or utilizingin designing MAPs or has utilizing been that MAPs the composition has been that of thethe innercomposition and outer of leaflets the inner of the and cell outer leafletsmembrane of the cell remains membrane static overtime. remains static The cell overtime. membrane, The however, cell membrane, contains however, enzymes that contains constantly enzymes that reshapeconstantly lipid reshape composition: lipid flippasescomposition: and flopases flippases [16 –and19]. flopases Organisms [16 that–19] can. Orga morenisms rapidly that alter can the more rapidlycomposition alter the ofcomposition their cell membrane of their cell leaflets membrane via mutated leaflets flippases via mutated can better flippases respond tocan a threatbetter from respond to a threatMAPs, from and hence, MAPs may, and develop hence,resistance. may develop Finally, resistance. when other Finally, small when drugs other do reach small the drugs membrane do reach the membraneor intracellular or intracellular space, they mayspace, be they expelled may bybe endogenousexpelled by transportersendogenous [20 transporters–22]. This is [20 already–22]. This is alreadywell accepted well accepted in the microbiology in the microbiology community community as an emerging as an avenue emerging of antimicrobial-resistance avenue of antimicrobial- resistancewherein wherein MAPs may MAPs prove may synergistic. prove synergistic. In this review, In this we argue review that, we to combatargue resistancethat to combat by flippases, resistance peptidases, charged structural components, and transporters, a method must be employed that looks by flippases, peptidases, charged structural components, and transporters, a method must be for potential drug synergies that could specifically address those three avenues. employed that looks for potential drug synergies that could specifically address those three avenues. FigureFigure 1. This 1. Thisfigure figure provides provides a graphical a graphical overview overview of the of theinformation information on onpossible possible methods methods by by which cellswhich may develop cells may resistance develop resistanceto MAPs tothrough MAPs protein through structures protein structures,, and hence and, targets hence, targetsfor synergistic for interactions.synergistic interactions. 2. Defining Antimicrobial Synergy and Resistance 2.1. Minimum Inhibitory Concentration Assays When discussing activity, it is important to try to define and quantify the terms synergy and resistance with respect to antimicrobial peptides. The common metric for comparing antimicrobial Antibiotics 2020, 9, 620 3 of 15 2. Defining Antimicrobial Synergy and Resistance 2.1. Minimum Inhibitory Concentration Assays When discussing activity, it is important to try to define and quantify the terms synergy and resistance with respect to antimicrobial peptides. The common metric for comparing antimicrobial efficacy