Towards Development of Small Molecule Lipid II Inhibitors as Novel Antibiotics

RESEARCH ARTICLE Towards Development of Small Molecule Lipid II Inhibitors as Novel Antibiotics Jamal Chauhan1,3, Steven Cardinale4, Lei Fang1,2,3, Jing Huang1,2, Steven M. Kwasny4, M. Ross Pennington5, Kelly Basi5, Robert diTargiani5, Benedict R. Capacio5, Alexander D. MacKerell, Jr1,2, Timothy J. Opperman4, Steven Fletcher1, Erik P. H. de Leeuw6* a11111 1 Department of Pharmaceutical Sciences, University of Maryland, School of Pharmacy, Baltimore, Maryland, United States of America, 2 Computer-Aided Drug Design Center, University of Maryland, School of Pharmacy, Baltimore, Maryland, United States of America, 3 Center for Biomolecular Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 4 Microbiotix, Inc., One Innovation Drive, Worcester, Massachusetts, United States of America, 5 U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland, United States of America, 6 Institute of Human Virology & Department of Biochemistry and Molecular Biology of the University of Maryland Baltimore School of Medicine, Baltimore, Maryland, United States of America * OPEN ACCESS Citation: Chauhan J, Cardinale S, Fang L, Huang J, Kwasny SM, Pennington MR, et al. (2016) Towards Development of Small Molecule Lipid II Inhibitors as Novel Antibiotics. PLoS ONE 11(10): e0164515. doi:10.1371/journal.pone.0164515 Editor: Hendrik W. van Veen, University of Cambridge, UNITED KINGDOM Received: June 13, 2016 Accepted: September 25, 2016 Published: October 24, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Abstract Recently we described a novel di-benzene-pyrylium-indolene (BAS00127538) inhibitor of Lipid II. BAS00127538 (1-Methyl-2,4-diphenyl-6-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)pyryl-1-ium) tetrafluoroborate is the first small molecule Lipid II inhibitor and is structurally distinct from natural agents that bind Lipid II, such as vancomycin. Here, we describe the synthesis and biological evaluation of 50 new analogs of BAS001 27538 designed to explore the structure-activity relationships of the scaffold. The results of this study indicate an activity map of the scaffold, identifying regions that are critical to cytotoxicity, Lipid II binding and range of anti-bacterial action. One compound, 6jc48-1, showed significantly enhanced drug-like properties compared to BAS00127538. 6jc48-1 has reduced cytotoxicity, while retaining specific Lipid II binding and activity against Enterococcus spp. in vitro and in vivo. Further, this compound showed a markedly improved pharmacokinetic profile with a half-life of over 13 hours upon intravenous and oral administration and was stable in plasma. These results suggest that scaffolds like that of 6jc48-1 can be developed into small molecule antibiotic drugs that target Lipid II. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by NIH grant AI092033 (www.nih.gov), CBT grant PR155EDL1 (www.medschool.umaryland.edu/cbt/), a Maryland Innovation Initiative (http://tedco.md/program/themaryland-innovation-initiative-mii/) award and a UM Ventures seed grant (www.umventures.org) to EdL. Further support was provided through the University of Maryland Computer Aided Drug Design Center and School of Pharmacy. The funders had no role in study design, data collection Introduction The biosynthesis pathway of the bacterial cell wall is well studied and a validated target for the development of antibacterial agents. Cell wall biosynthesis involves two major processes; 1) the biosynthesis of cell wall teichoic acids and 2) the biosynthesis of peptidoglycan. Key molecules in these pathways, including enzymes and precursor molecules are attractive targets for the development of novel antibacterial agents [1–4]. The cell wall of both Gram-negative and Gram-positive bacteria comprises a peptidoglycan layer which is composed of a polymer of alternating amino sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid PLOS ONE | DOI:10.1371/journal.pone.0164515 October 24, 2016 1 / 19 Lipid II Inhibitors as Novel Antibiotics and analysis, or preparation of the manuscript. Microbiotix Inc. provided support in the form of salaries for authors [SC, SK and TO], but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors of this manuscript have the following competing interests: US patent # 8,796,323 (Defensin-like molecules as novel antimicrobial agents) (AM and EdL, inventors); US nonprovisional patent application # 13/911,234 (Defensin-like molecules as novel antimicrobial agents) (AM and EdL, Inventors), US Provisional Patent application #62/270,184 (Small Molecule Lipid II inhibitors as novel antimicrobial agents) (JH, SF, AM and EdL, inventors). SC, SK and TO are paid employees at Microbiotix, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. (MurNAc). On the cytoplasmic side of the plasma membrane, the soluble precursor UDPMurNAc-pentapeptide is linked to the membrane carrier bactoprenol- phosphate (C55P) yielding Lipid I. In a second step GlcNac is added by the enzyme MurG to yield Lipid II [1]. Lipid II is essential for cell wall biosynthesis, is synthesized in limited amounts and has a high turnover rate, making it an attractive and established target for antibacterial compounds. Various classes of natural antibiotic peptides have been discovered that bind Lipid II, including depsipeptides, lantibiotics, cyclic peptides and glycopeptides [1]. Of these, vancomycin and its more recently developed derivatives daptomycin, oritavancin and telavancin are approved as first line treatments for Gram-positive infections [5–8]. However, resistance to these drugs is increasingly reported [9–11]. Several studies on defensins, effector peptides of innate immunity [12], revealed specific interactions with Lipid II, adding another class of natural compounds to the growing list of structurally unrelated peptides that bind this target [13– 19]. Based on the interaction between Lipid II and Human Neutrophil Peptide -1, we previously identified, for the first time, low molecular weight synthetic compounds that target Lipid II with high specificity and affinity [20]. One of our lead compounds, BAS00127538, was characterized further and revealed a unique interaction with Lipid II that differs from antibiotics currently in clinical use or development [20]. In this study, we report on the structural and functional relationships of derivatives of BAS00127538. Materials and Methods Materials and Bacterial St (...truncated)