Contents lists available at ScienceDirect BBA - Biomembranes journal homepage: www.elsevier.com/locate/bbamem Hydrophobic interactions modulate antimicrobial peptoid selectivity towards anionic lipid membranes Konstantin Andreev a,1 , Michael W. Martynowycz a,c,2 , Mia L. Huang b,3 , Ivan Kuzmenko c , Wei Bu d , Kent Kirshenbaum b , David Gidalevitz a, ⁎ a Department of Physics, Center for Molecular Study of Condensed Soft Matter (μCoSM), Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, 3440 South Dearborn Street, Chicago, IL 60616, United States b Department of Chemistry, New York University, 100 Washington Square East, New York, NY 10003, United States c Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, United States d The Center for Advanced Radiation Sources (CARS), University of Chicago, Chicago, IL 60637, United States ARTICLE INFO Keywords: Antimicrobial peptoids Lipid membranes Cytotoxicity AFM Hydrophobicity X-ray scattering ABSTRACT Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epi- fluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies de- monstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow different optimization schemes as com- pared to their natural analogues. 1. Introduction Antimicrobial peptides (AMPs) are a component of innate immunity that exhibit broad-spectrum antimicrobial activity [1,2]. AMPs target bacterial cells by electrostatic interactions between the positively charged peptide moieties and the negatively charged lipid headgroups [3,4]. The surface of nearly all bacteria has a net negative charge, while eukaryotic membranes are typically zwitterionic [5,6]. Selectivity is largely thought to be due to elementary electrostatics [7]. However, antimicrobial peptides are still capable of interacting with mammalian cells by permeating into the hydrophobic core of lipid bilayer [8]. Links between cytotoxicity and peptide structural differences are not well understood yet [9–11]. Larger hydrophobic content of an AMP en- hances its antimicrobial activity up to a critical level [12,13]. Further increases in net hydrophobicity are associated with the reduced se- lectivity [14,15]. For example, magainin-II displays bactericidal ac- tivity against Gram-negative species, whereas its analogues with higher numbers of non-polar moieties also inhibit growth of both Gram- positive bacteria and eukaryotic cells [16,17]. Comparative analysis of a novel family of synthetic AMPs composed of oligo-lysine sequences adjacent to an Ala/Trp/Phe linker shows two steps in membrane insertion [18]. The first step corresponds to the minimal hydrophobicity required for peptides to be transferred from the aqueous environment to the negatively charged lipid bilayer, whereas reaching the second critical point allows AMP to interact with neutral lipids [19]. Stark et al. demonstrated that by increasing the number of cationic residues the peptide sequence can be converted into a highly active amphipathic molecule [20]. If average hydrophobicity is above the second critical point, the peptides become capable of dis- rupting eukaryotic membranes [21]. Clinical application of AMPs has been unsuccessful [22,23]. De- veloping novel synthetic molecules that demonstrate high selectivity and activity is therefore crucial [24]. In poly-N-substituted glycines, or “peptoids”, the amino acid side-chains are linked to the peptide back- bone trough amide nitrogen rather than the α-carbon [25,26]. Peptoids display high antimicrobial efficacy in vitro and in vivo [27–29] https://doi.org/10.1016/j.bbamem.2018.03.021 Received 8 December 2017; Received in revised form 15 March 2018; Accepted 26 March 2018 ⁎ Corresponding author. 1 Present addresses: Howard Hughes Medical Institute, Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208 (USA). 2 Present addresses: Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147 (USA). 3 Present addresses: Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA, 92093-0358 (USA). E-mail address: gidalevitz@iit.edu (D. Gidalevitz). BBA - Biomembranes 1860 (2018) 1414–1423 Available online 03 April 2018 0005-2736/ © 2018 Elsevier B.V. All rights reserved. T