Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

“…Foot-and-mouth disease, a disease of cloven-hoofed livestock, exacts a severe economic toll on affected communities. 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.…”

“…between trypsin or chymotrypsin and the cyclotide was determined by using enzyme concentrations lower than the K i . Thus, proteases (0.005 nM, 50 µL trypsin or 0.5 nM, 50 µL chymotrypsin, 12.5 µM, 50 µL subtilisin and 1 nM, 50 µL thrombin) were incubated at a suitable range of concentrations 20 of inhibitor in TBS for 1 hour. The measurement was started by the addition of substrate: (5 µM, 100 µL N-p-tosyl-GlyPro-Arg-AMC (AMC: 7-amido-4-methyl coumarin) for trypsin and thrombin assays, or succinimidyl-Ala-Ala-ProPhe-AMC (10 µM, 100 µL for chymotrypsin or 19 µM, 25 100 µL for subtilisin assays).…”

“…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.…”

“…In addition to the naturally-occurring MCoTI-I and MCoTI-II cyclotides, a focussed series of MCoTI-II analogues bearing 15 alternative residues in place of Lys10 was designed to enable a systematic investigation of the influence of this position on protease inhibition. 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.…”

“…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.…”