Novel antibiotics: C-2 symmetrical macrocycles inhibiting Holliday junction DNA binding by E. coli RuvC Po-Shen Pan, a,  Fiona A. Curtis, b,  Chris L. Carroll, a Irene Medina, a Lisa A. Liotta, a Gary J. Sharples b, * and Shelli R. McAlpine a, * a Department of Chemistry and Biochemisty, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA b Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen’s Campus, Stockton-on-Tees TS17 6BH, UK Received 28 February 2006; revised 10 March 2006; accepted 15 March 2006 Available online 11 April 2006 Abstract—Holliday junctions (HJs) are formed as transient DNA intermediates during site-specific and homologous recombination. Both of these genetic exchange pathways are critical for normal DNA metabolism and repair. Trapping HJs leads to bacterial cell death by preventing proper segregation of the resulting interlinked chromosomes. Macrocyclic peptides designed to target this inter- mediate were synthesized with the goal of identifying compounds with specificity for this unique molecular target. We discovered ten macrocycles, both hexameric and octameric peptides, capable of trapping HJs in vitro. Those macrocycles containing tyrosine res- idues proved most effective. These data demonstrate that C-2 symmetrical macrocycles offer excellent synthetic targets for the devel- opment of novel antibiotic agents. Furthermore, the active compounds identified provide valuable tools for probing different pathways of recombinational exchange. Ó 2006 Elsevier Ltd. All rights reserved. 1. Introduction The emergence of new microbial pathogens coupled with a dramatic rise in the incidence of drug resistance poses a considerable challenge to human health. 1–3 We face the prospect of a post-antibiotic era where relatively minor hospital procedures can lead to life-threatening, untreatable infections. In order to be effective, we need new antibiotics that target unique sites in resistant strains of bacteria. The Holliday junction (HJ), a four- stranded joint (Fig. 1) derived from the recombinational exchange of DNA chains during site-specific and homol- ogous recombination reactions, 4 presents a potential target for a new spectrum of antimicrobials. Recombi- national processes are vital for accurate chromosomal segregation and DNA repair, the salvage of stalled rep- lication forks, and generating the rearrangements that fuel evolution. 5 Blocking recombination reactions by trapping the HJ intermediate prevents transmission of 0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2006.03.028 Keywords: Cyclicpeptides; Macrocycles; Antibiotics; Holliday junc- tion; Antibiotic resistance; Peptides. * Corresponding authors. Tel.: +1 619 594 5580 (S.R.M.); e-mail: mcalpine@sciences.sdsu.edu   These authors contributed equally to this work. Figure 1. Model of Escherichia coli RuvC protein bound to a square planar Holliday junction. The DNA is shown in yellow and the crystal structure of homodimeric RuvC in green. Residues important for catalysis (Asp-7, Glu-66, Asp-138, and Asp-141) are highlighted in red. RuvC is thought to bind this HJ conformation during branch migration as part of a RuvABC complex. Bioorganic & Medicinal Chemistry 14 (2006) 4731–4739