Discovery and target identification of SICLOPPS-derived antibacterial cyclic peptides

Abstract

The rise and rapid spread of antibiotic resistance is one of the great challenges facing modern medicine, creating an urgent need for novel and innovative antibiotics. Synthetic small molecule screens have historically yielded few antibiotics used clinically, but synthetic cyclic peptide libraries may revolutionise antibiotic discovery, as they can engage a greater breadth of challenging targets than small molecule compounds. Split-intein circular ligation of peptides and proteins (SICLOPPS) is a method of generating genetically encoded cyclic peptide libraries which can be expressed and screened intracellularly. In this work, a 3.2-million-membered CXXXXX library (where X = any of the 20 canonical amino acids) was created using SICLOPPS and screened for inhibitors of the bacterial Rod system. The Rod system contains both a validated antibiotic target and potential new targets and is an attractive focus for antibiotic discovery. The Rod system is essential in E. coli and other rod-shaped bacteria, but not in E. coli exhibiting upregulation of the cell division protein FtsZ (termed FtsZUP cells). Mecillinam is an antibiotic which causes toxic malfunction of the Rod system. Therefore, Rod system inhibition confers mecillinam resistance in FtsZUP E. coli but is lethal to wild-type E. coli. The CXXXXX library was screened in FtsZUP E. coli DH5α for cyclic peptides which suppress mecillinam toxicity. NGS analysis and motif discovery identified 13 cyclic peptides of interest from the screen. One of these cyclic peptides, cyclo-CVKYKP, was found to cause modest growth inhibition of E. coli DH5α. Cyclo-CVKYKP rescued growth of FtsZUP E. coli DH5α treated with mecillinam and failed to block the growth of FtsZUP E. coli DH5α in which the Rod system is non-essential. Scanning electron microscopy found that cyclo-CVKYKP-treated FtsZUP E. coli DH5α cells exhibit morphological defects and frequently grow as spheres instead of rods. These observations indicate that cyclo-CVKYKP is a Rod system inhibitor.Cyclo-SMDIKG is a previously reported SICLOPPS-derived antibacterial cyclic peptide with an unknown mode of action. To shed light on its mode of action, eight E. coli proteins were tested for their ability to suppress cyclo-SMDIKG toxicity when overexpressed. This led to the identification of ErpA, an iron-sulfur cluster protein essential for aerobic and anaerobic respiration in E. coli, as a candidate cyclo-SMDIKG target. Microscale thermophoresis (MST) analysis of cyclo-SMDIKG binding to apo-ErpA found a likely very weak binding interaction. However, MST analysis of cyclo-SMDIKG binding to holo-ErpA suggested preferential binding to holo-ErpA with much higher affinity. Further work is needed to confirm this observation.This study showcases SICLOPPS as a promising and flexible approach to synthetic library screening for antibiotic discovery, yielding antibacterial cyclic peptides with putative novel targets to guide future innovative antibiotic development.<br/