"Splicing up" drug discovery. Cell-based expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides

Abstract

The present paper reviews the use of protein splicing for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes thus providing a new way for finding therapeutic agents.

Figure 1

Backbone cyclization of polypeptides using native chemical Ligation. A. Principle of Native Chemical Ligation (NCL). B. Intramolecular NLC leads to the formation of a backbone cyclized polypeptide.

Figure 2

Biosynthesis of recombinant polypeptide α-thioesters. A. Scheme representing the proposed canonical mechanism for protein splicing mediated by a Cys-intein. B. Expression and purification of recombinant polypeptide thioesters using a modified intein fusion protein. In the modified intein (represented with an asterisk) the last Asn residue of the intein has been mutated to Ala to prevent C-terminal cleavage and splicing. This mutation allows trapping a thioester intermediate that can be cleaved with a thiol to provide the corresponding thioester funtion.

Figure 3

Biosynthetic approach for the in vivo production of cyclotides inside live E. coli cells. Backbone cyclization of the linear cyclotide precursor is mediated by a modified protein splicing unit or intein. The cyclized product then folds spontaneously in the bacterial cytoplasm.

Figure 4

Production of backbone cyclized polypeptides using protein trans-splicing. A. Scheme representing the proposed canonical mechanism for protein trans-splicing mediated by a split Cys-intein. B. Cyclization of polypeptides using protein trans-splicing. To facilitate cyclization, the N-intein and C-intein moieties are fused the C- and N-terminus of the polypeptide to be cyclized, respectively.

Figure 5

Biosysnthesis of cyclic polypeptides using intramolecular sortase-mediated ligation. A. Principle of sortase-mediated ligation. Sortase A first recognizes an LPXTG sequence within polypeptide 1 and cleaves the amide bond between the Thr and the Gly with an active-site Cys184, generating a covalent acyl-enzyme intermediate. The thioester intermediate is then attacked by an amino group of the oligo Gly-containing polypeptide 2, which allows the ligation of the two polypeptides by a native peptide bond. B. Polypeptide cyclization of a dual tagged polypeptide by an intra-molecular transpeptidation reaction.

Figure 6

Shematic representation of the putative mechanism of protease-catalysed cyclotide cyclization. Cyclotides are a large family of plant defence proteins characterized by a cystine knot and cyclic backbone. Their prototypic linear precursor protein (top) comprises an endoplasmic reticulum (ER)-targeting sequence (dark blue), a pro-region (purple), an N-terminal repeat region (Ntr; green), a cyclotide domain (light grey) and a C-terminal tail (red). It features also a conserved asparagine (yellow) at the C-terminal cleavage point of the cyclotide domain. The precursor is processed in the ER and vacuole: disulfide bonds are formed (yellow) and a range of unidentified proteases (brown) trim the precursor. In the final stage the active-site cysteine of an AEP (yellow) displaces the C-terminal tail to form an enzyme-acyl intermediate (boxed). This intermediate is then attacked by the cyclotide N-terminal glycine to form the mature cyclic peptide. Figure adapted from reference [60].

Scheme 1

Summary of the technologies used for the biosynthesis of backbone cyclized peptides. All these methods rely on the ribosomal synthesis of protein precursors that undergo protein splicing mediated by inteins, proteases or sortases. Three examples of naturally occurring backbone cyclized peptides with potential therapeutic value are also shown in the middle of the scheme. MCoTI-II is a naturally occurring cyclotide with trypsin inhibitory activity found in the seeds of tropical squash (Momordica conchichinensis) [74, 75] (PDB entry: 1IB9). SFTI-1 is a Bowman-Birk protease inhibitor found in the seeds of sunflower (Helianthus annuus) [76, 77] (PDB entry: 1JBN). Both peptides have been biosynthesized using EPL [49]. RTD-1 is a primate defensin with strong antibacterial and antiviral activity [78, 79] (PDB entry: 1HVZ).