Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies

Abstract

Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets.

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis

“…Cyclotides have attracted significant interest as molecular scaffolds for the development of peptide-based therapeutics due to their pharmaceutically relevant chemical and biological properties. − First, their cyclic cystine knot (CCK) framework, which comprises a cyclic head-to-tail backbone with a knotted disulfide core, confers cyclotides with exceptional physical, chemical, and biological stability . Second, cyclotides have a wide range of biological activities including uterotonic, anti-HIV, − antimicrobial, antifouling, hemolytic, , and cytotoxic activities − as well as protease inhibition, , and immunosuppressive properties . Third, cyclotides have promising druglike properties including cell-permeability , and oral activity. , Indeed, their clinical potential is highlighted by the cyclotide [T20K]­kalata B1, which is currently undergoing Phase 1 clinical trials for oral delivery in the treatment of multiple sclerosis .…”

Design of Hepatitis C Protease Inhibitors Based on Knottin Fold

“…These residues are located in the loop for which a successful loop grafting was done [11b,13,36] . In vitro tests of such modified knottins showed that these changes did not destroy the knottin structure [11b] and new chimeric knottins showed an increased potency and selectivity [13,36] . Cys8 was not changed because it was a part of the cystine knot.…”

“…Val7 was changed to Leu, Pro9 to Tyr and Lys10 to Leu (for clarity here and below the knottin residues are underlined. These residues are located in the loop for which a successful loop grafting was done [11b,13,36] . In vitro tests of such modified knottins showed that these changes did not destroy the knottin structure [11b] and new chimeric knottins showed an increased potency and selectivity [13,36] .…”

Mutagenesis of cyclotide Cter 27 exemplifies a robust folding strategy for bracelet cyclotides

“…Cyclotides have a wide range of biological activities, including uterotonic, [ 10 ] anti‐HIV, [ 11–13 ] antimicrobial, [ 14 ] antifouling, [ 15 ] hemolytic, [ 16,17 ] and cytotoxic activities. [ 18–20 ] Cyclotides have also demonstrated protease inhibition, [ 21,22 ] and immunosuppression properties, [ 23 ] as well as desirable drug‐like properties such as cell permeability [ 24,25 ] and oral activity. [ 26,27 ] Several cyclotide‐based peptides have been shown to be orally active, including kalata B1 grafted with bradykinin antagonist peptides for inflammatory pain treatment [ 26 ] and [T20K] kalata B1, which is being developed as a treatment for multiple sclerosis.…”