5th International Conference on Advances in Chemistry and Applied Chemistry (ICACAC-5) OL 8: The manipulation of "SICLOPPS" (Split Intein Circular Ligation of Peptides and Proteins) technology to identify cyclic peptide inhibitors of a novel pathway in E.coli S.A. El-Mowafi Peptide Chemistry Department, National Research Center, Cairo Egypt, Email: [email protected] Abstract The assembly of molecular libraries presents an important source of candidates for identification of new biological targets. Recently, large libraries of cyclic peptides named “SICLOPPS” have been produced within bacterial E. coli cells, whereas head-to-tail cyclization of the target sequence occurs spontaneously in vivo. A SICLOPPS library is exemplified by the general formula Ser-Gly-Trp-X5, where X represents one of the 20 essential amino acids (1,2). Herein, we describe the use of the described library, in conjunction with a reporter strain, in which the expression of yellow fluorescent protein (YFP) was engineered, in order to perform a genetic screen. This screen identified cyclic peptide inhibitors of a novel regulatory pathway, whose components play a critical role in the maintenance of the cell envelope and are required for the virulence in several Gram-negative pathogens. These candidates were, subsequently, chemically synthesized and the Organized by Chemical Industries Research Division-National Research CentreDokki, Cairo, Egypt 1 5th International Conference on Advances in Chemistry and Applied Chemistry (ICACAC-5) biochemistry was optimized to determine their molecular targets. The identification and validation of these inhibitors, thus, provide a strategy for the development of novel generations of potent antibiotics, via new mechanisms of action. The realized results will be presented and discussed in details (3,4). Keywords: SICLOPPS, cyclic peptides, genetic screen, virulence. References: 1. Scott CP, Abel-Santos E, Wall M, Wahnon DC, Benkovic SJ. 1999. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. USA. 96:13638–13643. 2. Tavassoli A, Benkovic SJ. 2007. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc. 2:1126–1133. 3. El-Mowafi SA, Alumasa JN, Ades SE, Keiler KC. 2014. CellBased Assay to Identify Inhibitors of the Hfq-sRNA Regulatory Pathway. Antimicrob. Agents Chemother. 58: 5500- 5509. 4. El-Mowafi SA, Sineva EV, Alumasa JN, Nicoloff H, Tomsho JW, Ades SE, Keiler KC. 2014. Identification of Inhibitors of a Bacterial Sigma Factor Using a New High-throughput Screening Assay. Antimicrob. Agents Chemother. (Submitted) Organized by Chemical Industries Research Division-National Research CentreDokki, Cairo, Egypt 2