Rational design and selection of next-generation cyclotide drugs using the knotted cyclic peptide scaffold MCoTI-II
Sven Ullrich
A # , Grégoire Jean-Baptiste Philippe A # , Choi Yi Li A # and Hiroaki Suga A *
A
# These authors contributed equally and share first authorship.
Handling Editor: Ed Nice
Abstract
Naturally occurring cyclic peptides have emerged as intriguing and relevant scaffolds for next-generation drug design. Especially peptides from the highly constrained cyclotide family provide an exceptional opportunity for drug discovery, given their characteristic head-to-tail cyclic structure and cystine-knotted core that confer favourable pharmaceutical properties. As a result, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II), a cyclic knottin peptide natural product of gấc fruit, has been the centre of various molecular engineering efforts. To target medicinally relevant proteins, surface-exposed loops of the mini-protein have been purposefully modified or entirely selected de novo to install bioactive epitopes. This mini-review aims to provide a succinct overview of recent studies on MCoTI-II-derived structures in inhibitor and diagnostic design. A variety of methods facilitate access to MCoTI-II-based next-generation therapeutics and diagnostics, including rational design, loop grafting and genetically encoded discovery platforms.
Keywords: biologics, cyclotides, display screenings, grafting, knottins, natural products, next-generation drugs, peptides, proteins, rational design.
References
1 Hernandez JF, Gagnon J, Chiche L, Nguyen TM, Andrieu JP, Heitz A, et al. Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry 2000; 39: 5722-5730.
| Crossref | Google Scholar | PubMed |
2 Chiche L, Heitz A, Gelly JC, Gracy J, Chau PT, Ha PT, et al. Squash inhibitors: from structural motifs to macrocyclic knottins. Curr Protein Pept Sci 2004; 5: 341-349.
| Crossref | Google Scholar | PubMed |
3 de Veer SJ, Kan M-W, Craik DJ. Cyclotides: from structure to function. Chem Rev 2019; 119: 12375-12421.
| Crossref | Google Scholar | PubMed |
4 Do TVT, Fan L, Suhartini W, Girmatsion M. Gấc (Momordica cochinchinensis) fruit: a functional food and medicinal resource. J Funct Foods 2019; 62: 103512.
| Crossref | Google Scholar |
5 Phan-Thi H, Waché Y. Behind the myth of the fruit of heaven, a critical review on gấc (Momordica cochinchinensis). Curr Med Chem 2019; 26: 4585-4605.
| Crossref | Google Scholar | PubMed |
6 Kan M-W, Craik DJ. Discovery of cyclotides from Australasian plants. Aust J Chem 2020; 73: 287-299.
| Crossref | Google Scholar |
7 Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Le Nguyen D, Sommerhoff CP, Diederichsen U, et al. Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II. Biol Chem 2005; 386: 1301-1306.
| Crossref | Google Scholar | PubMed |
8 Heitz A, Hernandez JF, Gagnon J, Hong TT, Pham TT, Nguyen TM, et al. Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins. Biochemistry 2001; 40: 7973-7983.
| Crossref | Google Scholar | PubMed |
9 Mylne JS, Chan LY, Chanson AH, Daly NL, Schaefer H, Bailey TL, et al. Cyclic peptides arising by evolutionary parallelism via asparaginyl–endopeptidase-mediated biosynthesis. Plant Cell 2012; 24: 2765-2778.
| Crossref | Google Scholar | PubMed |
10 Tyler TJ, Durek T, Craik DJ. Native and engineered cyclic disulfide-rich peptides as drug leads. Molecules 2023; 28: 3189.
| Crossref | Google Scholar | PubMed |
11 Xie J, Kan MW, de Veer SJ, Wang C, Craik DJ. Display technologies for expanding the pharmaceutical applications of cyclotides. Isr J Chem 2024; 64: e202400010.
| Crossref | Google Scholar |
12 Colgrave ML, Craik DJ. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 2004; 43: 5965-5975.
| Crossref | Google Scholar | PubMed |
13 Chan LY, Gunasekera S, Henriques ST, Worth NF, Le S-J, Clark RJ, et al. Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds. Blood 2011; 118: 6709-6717.
| Crossref | Google Scholar | PubMed |
14 Quimbar P, Malik U, Sommerhoff CP, Kaas Q, Chan LY, Huang YH, et al. High-affinity cyclic peptide matriptase inhibitors. J Biol Chem 2013; 288: 13885-13896.
| Crossref | Google Scholar | PubMed |
15 Swedberg JE, Ghani HA, Harris JM, de Veer SJ, Craik DJ. Potent, selective, and cell-penetrating inhibitors of kallikrein-related peptidase 4 based on the cyclic peptide MCoTI-II. ACS Med Chem Lett 2018; 9: 1258-1262.
| Crossref | Google Scholar | PubMed |
16 Laskowski M, Qasim MA. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim Biophys Acta 2000; 1477: 324-337.
| Crossref | Google Scholar | PubMed |
17 Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. Nat Rev Drug Discov 2021; 20: 309-325.
| Crossref | Google Scholar | PubMed |
18 Katoh T, Goto Y, Reza MS, Suga H. Ribosomal synthesis of backbone macrocyclic peptides. Chem Commun 2011; 47: 9946-9958.
| Crossref | Google Scholar | PubMed |
19 Henriques ST, Craik DJ. Cyclotides as templates in drug design. Drug Discov Today 2010; 15: 57-64.
| Crossref | Google Scholar | PubMed |
20 de Veer SJ, Weidmann J, Craik DJ. Cyclotides as tools in chemical biology. Acc Chem Res 2017; 50: 1557-1565.
| Crossref | Google Scholar | PubMed |
21 Camarero JA. Cyclotides, a versatile ultrastable micro-protein scaffold for biotechnological applications. Bioorg Med Chem Lett 2017; 27: 5089-5099.
| Crossref | Google Scholar | PubMed |
22 Qu H, Smithies BJ, Durek T, Craik DJ. Synthesis and protein engineering applications of cyclotides. Aust J Chem 2017; 70: 152-161.
| Crossref | Google Scholar |
23 Mishra M, Singh V, Tellis MB, Joshi RS, Singh S. Repurposing the MCoTI-II rigid molecular scaffold in to inhibitor of ‘papain superfamily’ cysteine proteases. Pharmaceuticals 2020; 14: 7.
| Crossref | Google Scholar | PubMed |
24 Felizmenio-Quimio ME, Daly NL, Craik DJ. Circular proteins in plants. J Biol Chem 2001; 276: 22875-22882.
| Crossref | Google Scholar | PubMed |
25 Huang Y-H, Jiang Z, Du Q, Yap K, Bigot A, Kaas Q, et al. Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1. J Biol Chem 2024; 300: 105682.
| Crossref | Google Scholar | PubMed |
26 Li CY, Rehm FBH, Yap K, Zdenek CN, Harding MD, Fry BG, et al. Cystine knot peptides with tuneable activity and mechanism. Angew Chem Int Ed 2022; 61: e202200951.
| Crossref | Google Scholar | PubMed |
27 Tian S, Durek T, Wang CK, Zdenek CN, Fry BG, Craik DJ, et al. Engineering the cyclization loop of MCoTI-II generates targeted cyclotides that potently inhibit factor XIIa. J Med Chem 2022; 65: 15698-15709.
| Crossref | Google Scholar | PubMed |
28 Sommerhoff CP, Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Diederichsen U, Kolmar H. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta. J Mol Biol 2010; 395: 167-175.
| Crossref | Google Scholar | PubMed |
29 Thongyoo P, Bonomelli C, Leatherbarrow RJ, Tate EW. Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J Med Chem 2009; 52: 6197-6200.
| Crossref | Google Scholar | PubMed |
30 Pinilla C, Appel JR, Blanc P, Houghten RA. Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. Biotechniques 1992; 13: 901-905.
| Google Scholar | PubMed |
31 Zimmerman M, Yurewicz E, Patel G. A new fluorogenic substrate for chymotrypsin. Anal Biochem 1976; 70: 258-262.
| Crossref | Google Scholar | PubMed |
32 Swedberg JE, Li CY, de Veer SJ, Wang CK, Craik DJ. Design of potent and selective cathepsin G inhibitors based on the sunflower trypsin inhibitor-1 scaffold. J Med Chem 2017; 60: 658-667.
| Crossref | Google Scholar | PubMed |
33 Swedberg JE, Nigon LV, Reid JC, de Veer SJ, Walpole CM, Stephens CR, et al. Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4. Chem Biol 2009; 16: 633-643.
| Crossref | Google Scholar | PubMed |
34 Swedberg JE, Mahatmanto T, Abdul Ghani H, de Veer SJ, Schroeder CI, Harris JM, et al. Substrate-guided design of selective FXIIa inhibitors based on the plant-derived Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) scaffold. J Med Chem 2016; 59: 7287-7292.
| Crossref | Google Scholar | PubMed |
35 Muratspahić E, Koehbach J, Gruber CW, Craik DJ. Harnessing cyclotides to design and develop novel peptide GPCR ligands. RSC Chem Biol 2020; 1: 177-191.
| Crossref | Google Scholar | PubMed |
36 Poth AG, Chan LY, Craik DJ. Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 2013; 100: 480-491.
| Crossref | Google Scholar | PubMed |
37 Chan LY, Craik DJ, Daly NL. Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy. Sci Rep 2016; 6: 35347.
| Crossref | Google Scholar | PubMed |
38 DeMarco SJ, Henze H, Lederer A, Moehle K, Mukherjee R, Romagnoli B, et al. Discovery of novel, highly potent and selective β-hairpin mimetic CXCR4 inhibitors with excellent anti-HIV activity and pharmacokinetic profiles. Bioorg Med Chem 2006; 14: 8396-8404.
| Crossref | Google Scholar | PubMed |
39 Aboye TL, Ha H, Majumder S, Christ F, Debyser Z, Shekhtman A, et al. Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity. J Med Chem 2012; 55: 10729-10734.
| Crossref | Google Scholar | PubMed |
40 Aboye T, Meeks CJ, Majumder S, Shekhtman A, Rodgers K, Camarero JA. Design of a MCoTI-based cyclotide with angiotensin (1-7)-like activity. Molecules 2016; 21: 152.
| Crossref | Google Scholar | PubMed |
41 Greenwood KP, Daly NL, Brown DL, Stow JL, Craik DJ. The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis. Int J Biochem Cell Biol 2007; 39: 2252-2264.
| Crossref | Google Scholar | PubMed |
42 Henriques ST, Lawrence N, Kan M-W, Malins LR, Craik DJ. Cell-penetrating cyclic and disulfide-rich peptides are privileged molecular scaffolds for intracellular targeting. Biochemistry 2025; 64: 1437-1449.
| Crossref | Google Scholar | PubMed |
43 D’Souza C, Henriques ST, Wang CK, Cheneval O, Chan LY, Bokil NJ, et al. Using the MCoTI-II cyclotide scaffold to design a stable cyclic peptide antagonist of SET, a protein overexpressed in human cancer. Biochemistry 2016; 55: 396-405.
| Crossref | Google Scholar | PubMed |
44 Huang YH, Henriques ST, Wang CK, Thorstholm L, Daly NL, Kaas Q, et al. Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold. Sci Rep 2015; 5: 12974.
| Crossref | Google Scholar | PubMed |
45 Huang YH, Chaousis S, Cheneval O, Craik DJ, Henriques ST. Optimization of the cyclotide framework to improve cell penetration properties. Front Pharmacol 2015; 6: 17.
| Crossref | Google Scholar | PubMed |
46 Ji Y, Majumder S, Millard M, Borra R, Bi T, Elnagar AY, et al. In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J Am Chem Soc 2013; 135: 11623-11633.
| Crossref | Google Scholar | PubMed |
47 Pazgier M, Liu M, Zou G, Yuan W, Li C, Li C, et al. Structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX. Proc Natl Acad Sci USA 2009; 106: 4665-4670.
| Crossref | Google Scholar | PubMed |
48 Philippe GJ-B, Huang Y-H, Mittermeier A, Brown CJ, Kaas Q, Ramlan SR, et al. Delivery to, and reactivation of, the p53 pathway in cancer cells using a grafted cyclotide conjugated with a cell-penetrating peptide. J Med Chem 2024; 67: 1197-1208.
| Crossref | Google Scholar | PubMed |
49 Lättig-Tünnemann G, Prinz M, Hoffmann D, Behlke J, Palm-Apergi C, Morano I, et al. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides. Nat Commun 2011; 2: 453.
| Crossref | Google Scholar | PubMed |
50 Herce HD, Schumacher D, Schneider AFL, Ludwig AK, Mann FA, Fillies M, et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nat Chem 2017; 9: 762-771.
| Crossref | Google Scholar | PubMed |
51 Sohrabi C, Foster A, Tavassoli A. Methods for generating and screening libraries of genetically encoded cyclic peptides in drug discovery. Nat Rev Chem 2020; 4: 90-101.
| Crossref | Google Scholar | PubMed |
52 Colas K, Bindl D, Suga H. Selection of nucleotide-encoded mass libraries of macrocyclic peptides for inaccessible drug targets. Chem Rev 2024; 124: 12213-12241.
| Crossref | Google Scholar | PubMed |
53 Ullrich S, Nitsche C. A RaPID response to SARS‐CoV‐2. Isr J Chem 2024; 64: e202300170.
| Crossref | Google Scholar |
54 Lopez-Morales J, Vanella R, Appelt EA, Whillock S, Paulk AM, Shusta EV, et al. Protein engineering and high‐throughput screening by yeast surface display: survey of current methods. Small Sci 2023; 3: 2300095.
| Crossref | Google Scholar | PubMed |
55 Jaroszewicz W, Morcinek-Orłowska J, Pierzynowska K, Gaffke L, Węgrzyn G. Phage display and other peptide display technologies. FEMS Microbiol Rev 2022; 46: fuab052.
| Crossref | Google Scholar | PubMed |
56 Huang Y, Wiedmann MM, Suga H. RNA display methods for the discovery of bioactive macrocycles. Chem Rev 2018; 119: 10360-10391.
| Crossref | Google Scholar | PubMed |
57 Lui BG, Salomon N, Wüstehube-Lausch J, Daneschdar M, Schmoldt H-U, Türeci Ö, et al. Targeting the tumor vasculature with engineered cystine-knot miniproteins. Nat Commun 2020; 11: 295.
| Crossref | Google Scholar | PubMed |
58 Kimura RH, Teed R, Hackel BJ, Pysz MA, Chuang CZ, Sathirachinda A, et al. Pharmacokinetically stabilized cystine knot peptides that bind alpha-V-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer. Clin Cancer Res 2012; 18: 839-849.
| Crossref | Google Scholar | PubMed |
59 Glotzbach B, Reinwarth M, Weber N, Fabritz S, Tomaszowski M, Fittler H, et al. Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1. PLoS ONE 2013; 8: e76956.
| Crossref | Google Scholar | PubMed |
60 Maaß F, Wüstehube-Lausch J, Dickgießer S, Valldorf B, Reinwarth M, Schmoldt HU, et al. Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4). J Pept Sci 2015; 21: 651-660.
| Crossref | Google Scholar | PubMed |
61 Liu W, de Veer SJ, Huang YH, Sengoku T, Okada C, Ogata K, et al. An ultrapotent and selective cyclic peptide inhibitor of human beta-factor XIIa in a cyclotide scaffold. J Am Chem Soc 2021; 143: 18481-18489.
| Crossref | Google Scholar | PubMed |
62 Gandini L, de Veer SJ, Chan CHH, Passmore MR, Liu K, Lundon B, Rachakonda R, White N, Rhodes M, Shanahan E, Yap K, See Hoe LE, Semenzin C, Li Bassi G, Fraser JF, Craik DJ, Suen JY, et al. Anticoagulation during extracorporeal membrane oxygenation (ECMO): a selective inhibitor of activated factor XII compared to heparin in an ex vivo model. ACS Pharmacol Transl Sci 2025; 8: 1260-1269.
| Crossref | Google Scholar | PubMed |
63 Xie J, Yap K, de Veer SJ, Edwardraja S, Durek T, Trau M, et al. High-throughput enrichment of functional disulfide-rich peptides by droplet microfluidics. Lab Chip 2025; 25(14): 3525-3536.
| Crossref | Google Scholar | PubMed |
64 Gründemann C, Stenberg KG, Gruber CW. T20K: an immunomodulatory cyclotide on its way to the clinic. Int J Pept Res Ther 2018; 25: 9-13.
| Crossref | Google Scholar |
