Harnessing cyclotides to design and develop novel peptide GPCR ligands

Abstract

Cyclotides are plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology that confers them with remarkable structural stability and resistance to proteolytic degradation. Recently, cyclotides have emerged as promising scaffold molecules for designing peptide-based therapeutics. Here, we provide examples of how engineering cyclotides using molecular grafting may lead to the development of novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. Integrating bioactive epitopes into stable cyclotide scaffolds can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. We also discuss and highlight the importance of engineered cyclotides as novel tools to study GPCR signaling.

Fig. 1. Cyclotide structure. NMR-derived three-dimensional structure and sequence of the (A) Möbius cyclotide kalata B1 (PDB: 1nb1), (B) bracelet cyclotide cycloviolacin O1 (PDB: 1nbj) and (C) trypsin inhibitor cyclotide MCoTI-II (PDB: 1ib9) are depicted. The cyclic cystine knot motif formed by three disulfide bonds is highlighted in yellow. The six cysteine residues are labeled I–VI, and the residues between adjacent cysteine residues represent loops 1–6. G₁ indicates the starting point of the sequence while a black arrow points to the direction of the peptide chain starting from N- to C-terminus. MCoTI-II = Momordica cochinchinensis trypsin inhibitor-II; PDB = protein data bank.

Fig. 2. Molecular targets and mechanism of action of cyclotides. l-Kalata B1 (purple, PDB: 1nb1) strongly interacts with the phosphatidylethanolamine-rich plasma membrane (straight arrow, left) whereas d-kalata B1's (blue, PDB: 2jue) interaction with the phosphatidylethanolamine-rich plasma membrane is significantly reduced (dashed arrow, left). By contrast, l-kalata B1 exhibits affinity towards a GPCR (straight arrow, right) but whether d-kalata B1 shows decreased receptor interaction remains to be determined (dashed arrow, right). Disulfide bonds are shown in yellow and phosphatidylethanolamine-rich plasma membrane and a GPCR are colored in red. GPCR = G protein-coupled receptor; PE = phosphatidylethanolamine; PDB = protein data bank.

Fig. 3. Examples of kB1 grafts. (A) A bioactive peptide epitope (orange) is inserted into loop 6 of kB1 (PDB: 1nb1) leading to stable peptide GPCR ligands with desired biological activity. Amino acid residues of native kB1 are shown in purple while grafted sequences are indicated in orange. Start and direction of the sequence is highlighted by a cysteine residue and a black arrow, respectively. Sequences and positions of bioactive epitopes grafted in loop 6 of kB1 discussed in this review are shown. Disulfide bonds or cysteine residues are highlighted in yellow and are labeled I–VI. Loops of cyclotides are labeled 1–6. (f) Represents the amino acid residue d-Phe. The starting point of the sequence has been taken from published studies. (B) Cartoon and surface overlay of kB1 and [GHFRWG; 23–28]kB1 (PDB: 2lur) as melanocortin pharmacophore highlighting the only key difference in the grafted loop 6. PDB = protein data bank; kB1 = kalata B1.

Fig. 4. Examples of cyclotide GPCR ligands grafted onto loop 6 of MCoTI-I scaffold (MCoTI-II NMR structure was used as template, PDB: 1ib9). Amino acid residues of native MCoTI-I are shown in green while grafted sequences are highlighted in orange. Start and direction of the sequence is indicated by a valine residue and a black arrow, respectively. The starting point of the sequence has been taken from published studies. Disulfide bonds or cysteine residues are shown in yellow and are labeled I–VI. B and p indicate the amino acid residues 2-naphthylalanine and d-Pro, respectively, while Z represents 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). X denotes citrulline residue except for MCo-AT1-7 where X is an l-2,3-diaminopropionic acid residue. The isopeptide bonds formed between the glycine residues and epitope are shown in red. MCoTI-I = Momordica cochinchinensis trypsin inhibitor-I; PDB = protein data bank.

Fig. 5. Summary of unresolved functions of engineered cyclotides as GPCR ligands. Native cyclotides have restricted capabilities to cross the blood–brain barrier (BBB), yet they are suitable to develop cyclotide grafts to target GPCRs in the central nervous system by penetrating the BBB. They may be harnessed as a tool to study biased signaling of GPCRs by either activating G protein-dependent or β-arrestin-dependent signaling pathways. Additionally, cyclotides may be able to initiate endosomal GPCR signaling of G_s protein-coupled receptors (G_s GPCR). For instance, following G_s GPCR activation by a grafted cyclotide, conformational change of a G_s GPCR leads to dissociation of the heterotrimeric G protein complex and production of cAMP (second messenger) via activation of adenylyl cyclase. While modulation of G protein-dependent pathways by cyclotides is well-studied (straight arrow), β-arrestin recruitment and thus β-arrestin-dependent pathways are poorly investigated (dashed arrows). Phosphorylation of a G_s GPCR by GRKs results in β-arrestin recruitment and initiation of clathrin-mediated endocytosis. According to a recent study published by Nguyen et al., once internalized, G_s GPCR and β-arrestin form a megaplex that activates G_s protein to enable a sustained endosomal G_s-mediated signaling. GRK = G protein-coupled receptor kinases, cAMP = cyclic adenosine monophosphate, G_s = stimulatory G protein, GPCR = G protein-coupled receptor.

Edin Muratspahić

Johannes Koehbach

Christian W. Gruber

David J. Craik