Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

Abstract

Perfect circle. We report the biosynthesis of a natively folded cyclotide, MCoTI‐II, in E. coli by intracellular backbone cyclization of a linear cyclotide–intein fusion precursor. The cyclized peptide then spontaneously folds into its native conformation. Biosynthetic access to correctly folded cyclotides allows the possibility of generating cell‐based combinatorial libraries that can be screened, inside living cells, for their ability to modulate or inhibit cellular processes.

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

“…56 In the work presented here, the first cyclotide 20 inhibitors of the foot-and-mouth viral 3C protease are reported, 48,57 based on a re-engineered MCoTI-II cyclotide scaffold. Though the potency of these first generation inhibitors is moderate (low μM), it is notable that cyclotides based on a trypsin inhibitor scaffold exhibit activity against 25 this cysteine protease. We anticipate that the efficient chemical and chemoenzymatic routes presented here will be readily adapted to ligation, screening and in situ purification of cyclotidebased libraries.…”

“…Boc-, 38 microwave-enhanced 24 and Fmoc- 14,30,34,35 based solid-phase peptide synthesis (SPPS) have all been used for cyclotide backbone construction, whilst recent work has shown that bacterial expression can also be an effective approach for 20 smaller scale production of the cyclotide backbone. 25,28 Backbone cyclisation may then be achieved by exposing a suitably side chain protected backbone peptide bearing an amino N-terminus and a carboxy C-terminus to standard peptide coupling conditions, but this approach suffers from 25 multiple drawbacks including poor peptide solubility and the need for high dilution. Thia-zip native chemical ligation (TZ-NCL), a technique first pioneered by the Tam group, 39,40 has emerged as the most effective method for backbone cyclisation of cysteine-rich cyclic peptides, and is particularly 30 applicable to the synthesis of cyclotides.…”

“…Lys10 is recognised as the key determinant of binding to trypsin by analogy to wellcharacterised homologous cystine-knot trypsin inhibitors such 20 as EETI, 29,43 and occupies the P 1 position in the substrate binding site. Engineering changes at this residue would therefore be expected to alter inhibitory activity against trypsin, and potentially enable the development of inhibitors of alternative proteases whose activity depends on the 25 presence of a residue other than Lys at P 1 , by analogy with previous work on peptide-based protease inhibitors. [44][45][46] Accordingly, open chain thioester MCoTI-II analogues (teMCoTI) bearing positively charged (Arg), hydrophobic (Val, Ala), aromatic (Phe) or amide (Gln) side chains were 30 synthesised (Scheme 2b), along with one analogue with an expanded active loop (Ala-Lys-Gln) to investigate the versatility of the synthetic strategy with a longer backbone.…”

“…Having established the inhibition activity and selectivity 25 profile of the MCoTI cyclotides, we next sought to design a simplified synthesis of the head-to-tail cyclic cystine-knot scaffold by utilising a biomimetic (chemoenzymatic) approach. It is known that related cyclic peptidic trypsin inhibitors such as BPTI 49,50 and SFTI-I 51 bind to the enzyme 30 active site in an inter-convertible mixture of non-covalent enzyme-peptide complex and covalently-bound peptidylenzyme, a non-productive analogue of the acyl-enzyme intermediate in proteolytic cleavage.…”

“…6,7 In contrast to many smaller naturally occurring cyclic peptides they are true gene products, and possess a highly stable cystine-knot † topology, 20 whereby two disulfide bridges form a ring through which a third is threaded. [8][9][10] This combination of intricate structure, diverse biological activity and unusual biogenesis has inspired a growing research effort directed towards understanding the synthesis and function of cyclotides both in the cell and in 25 vitro. 7,[11][12][13] In addition, their high resistance to degradation in vivo has attracted recent and widespread interest in the potential of cyclotides as scaffolds to present structured protein domains for therapeutic applications.…”