RESEARCH ARTICLE In Vivo, In Vitro, and In Silico Characterization of Peptoids as Antimicrobial Agents Ann M. Czyzewski 1☯ ,Håvard Jenssen 2,3☯ , Christopher D. Fjell 2 , Matt Waldbrook 2 , Nathaniel P. Chongsiriwatana 1 , Eddie Yuen 2 , Robert E. W. Hancock 2 *, Annelise E. Barron 1¤ * 1 Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, E136, Evanston, IL, 60208, United States of America, 2 Centre for Microbial Diseases and Immunity Research, #232–2259 Lower Mall Research Station, University of British Columbia, Vancouver, BC V6T 1Z4, Canada, 3 Dept. of Science, Systems & Models, Roskilde University, Universitetsvej 1, DK-4000, Roskilde, Denmark ☯ These authors contributed equally to this work. ¤ Current address: Department of Bioengineering, Stanford University, W300B James H. Clark Center, 318 Campus Drive, Stanford, CA, 94305–5444, United States of America * aebarron@stanford.edu (AEB); bob@hancocklab.com (REWH) Abstract Bacterial resistance to conventional antibiotics is a global threat that has spurred the devel- opment of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N- substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicro- bial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demon- strate the promising therapeutic potential of peptoids as antimicrobial agents. Introduction Drug development in the golden age of antibiotics (the 1960s and 1970s) resulted in an unprec- edented ability to control infections worldwide. However, initial successes bred a false sense of security that modern medicine could retain complete control over bacterial infections [1]. The emergence and re-emergence of multi-drug resistant (MDR) bacteria has since been recognized as an alarming threat to public health, and a dearth of novel antibiotic classes is creating PLOS ONE | DOI:10.1371/journal.pone.0135961 February 5, 2016 1 / 17 OPEN ACCESS Citation: Czyzewski AM, Jenssen H, Fjell CD, Waldbrook M, Chongsiriwatana NP, Yuen E, et al. (2016) In Vivo, In Vitro, and In Silico Characterization of Peptoids as Antimicrobial Agents. PLoS ONE 11 (2): e0135961. doi:10.1371/journal.pone.0135961 Editor: Mohamed N. Seleem, Purdue University, UNITED STATES Received: April 17, 2015 Accepted: January 14, 2016 Published: February 5, 2016 Copyright: © 2016 Czyzewski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: AEB acknowledges support from Northwestern University’s Institute for Bioengineering and Nanoscience in Advanced Medicine, the Dreyfus Foundation, a DuPont Young Investigator award, and NIH/NIAID Grant 5R01-AI072666. REWH gratefully acknowledges financial support from the Canadian Institutes for Health Research (CIHR) and the Foundation of the National Institutes of Health and CIHR through the Grand Challenges in Global Health Initiative. REWH is the recipient of a Canada Research Chair. AMC was supported by a 3M