SICLOPPS

(Split-Intein Circular Ligation of Peptides and Proteins)

The SICLOPPS methodology allows for the genetic encoding of libraries of small molecules, specifically cyclic peptides. Random peptide sequences are introduced into the libraries via oligonucleotide synthesis followed by PCR cloning. The PCR product is cloned into a plasmid that codes for a rearranged intein with an embeded random peptide sequence. After translation of the intein:peptide:intein fusion, the intein catalyzes the formation of a peptide bond between the first and last amino acid, thereby creating a cyclic peptide.

Circular ligation mechanism. An expressed fusion protein (F) folds to form an active protein ligase (1). The enzyme catalyzes an N-to-S acyl shift (2) at the target-IN junction to produce a thioester intermediate (T), which undergoes transesterification (3) with a side-chain nucleophile (X) at the IC-target junction to form a lariat intermediate (R). Asparagine side-chain cyclization (4) liberates the cyclic product as a lactone, and an X-to-N acyl shift (5) generates the thermodynamically favored, lactam product (O) in vivo.

The SICLOPPS and reverse two-hybrid technologies were integrated to pioneer a systematic method for discovering small-molecule modulators of protein-protein interactions. This method relies on linking protein complex formation to the expression of reporter genes, whose regulation can be monitored through chromogenic assays or host survival. Members of the SICLOPPS library that disrupt dimerization of the repressor, are selected through cell survival on selective media.

Schematic representation of the reverse two-hybrid system.

(A) The expression of protein fusions containing DNA-binding domains is induced, and they associate to repress a promoter that directs expression of three reporter genes: (i) HIS3, imidazole glycerol phosphate dehydratase; (ii) KanR (Kan), aminoglycoside 3'- phosphotransferase; (iii) lacZ, -galactosidase. The formation of protein complexes inhibits growth on minimal media.

(B) A cyclic peptide capable of inhibiting the protein-protein interaction rescues growth by inducing HIS3 and KanR expression.

The above system has been utilized for the discovery of novel inhibitors of several protein-protein interactions, such as AICAR transformylase, ribonucleotide reductase and HIV protease.

References:
	Horswill, A.R.; and Benkovic, S.J. “Identifying Small-Molecule Modulators of Protein-Protein Interactions”, in Current Protocols in Protein Science, John Wiley & Sons, Inc., pp. 19.15.1-19.15.19.
	Tavassoli, A.; Naumann, T.A.; and Benkovic, S.J. “Production of Cyclic Proteins and Peptides, in Nucleic Acids and Molecular Biology”, Vol. 16, Homing Endonucleases and Inteins, Marlene Belfort et al. (Eds.), Springer-Verlag Berlin Heidelberg, pp. 293-305.
	Naumann T.A; Savinov S.N; Benkovic S.J. “Engineering an affinity tag for genetically encoded cyclic peptides.” Biotechnol Bioeng. 2005 Dec 30;92(7):820-30.
	Horswill A.R.; Benkovic S.J. “Cyclic peptides, a chemical genetics tool for biologists.” Cell Cycle. 2005 Apr;4(4):552-5. Epub 2005 Apr 5. Review.
	Tavassoli, A.; Benkovic, S.J. “Genetically selected cyclic-peptide inhibitors of AICAR transformylase homodimerization.” Angew Chem Int Ed Engl. 2005 Apr 29;44(18):2760-3.
	Horswill, A.R.; Savinov, S.N. “A systematic method for identifying small-molecule modulators of protein-protein interactions.” Proc Natl Acad Sci USA. 2004 Nov 2;101(44):15591-6.
	Abel-Santos, E.; Scott, C.P.; Benkovic, S. J. “Use of inteins for the in vivo production of stable cyclic peptide libraries in E. coli.” Methods Mol Biol. 2003;205:281-94.
	Scott, C. P.; Abel-Santos, E.; Jones, A.D.; Benkovic, S. J. “Structural requirements for the biosynthesis of backbone cyclic peptide libraries.” Chem Biol. 2001 Aug;8(8):801-15.
	Scott, C. P.; Abel-Santos, E.; Wall, M.; Wahnon, D.C.; Benkovic, S.J. “Production of cyclic peptides and proteins in vivo.” Proc Natl Acad Sci USA. 1999 Nov 23;96(24):13638-43.