The weakness of ordinary peptides

Most peptides have a serious weakness as medicines. The body is full of enzymes called proteases whose job is to chop peptides and proteins into pieces. A typical linear peptide injected into the bloodstream may last only minutes. Swallowed, it is usually destroyed in the gut.

Nature, it turns out, has already solved this problem. Chapter 6 of Peptide Synthesis and Applications, from David Craik's laboratory at the University of Queensland, describes a family of peptides so tough they have become one of the most exciting scaffolds in peptide drug design.

Cyclotides

The chapter's main focus is cyclotides, peptides that plants make as a defence against pests. They have two features that, combined, make them extraordinary.

1. A circular backbone. Instead of having two free ends, the chain is joined head to tail, forming a ring. There are no loose ends for enzymes that nibble from the ends to grab.

2. A cystine knot. Inside the ring are six cysteines forming three disulfide bridges. They connect in a specific pattern (cysteine 1 to 4, 2 to 5, and 3 to 6) that creates a knot: one bridge threads through a loop formed by the other two and the backbone. The chapter calls this the cyclic cystine knot motif.

The chapter states the result plainly: this motif provides extraordinary stability. Cyclotides are known to resist heat, harsh chemicals and digestive enzymes that would destroy most peptides.

The circular backbone divides the molecule into six loops between the cysteines, and those loops can vary in sequence without breaking the knot.

Möbius and bracelet

The chapter describes two main cyclotide subfamilies, with names that reflect their geometry.

  • Möbius cyclotides, like the prototype kalata B1, contain a single twist in the backbone (a cis proline bond), like a Möbius strip.
  • Bracelet cyclotides have no twist, forming a simple ring.

A third group, trypsin-inhibiting peptides from the tropical plant Momordica cochinchinensis (MCoTI-I and MCoTI-II), share the ring and the knot but have unrelated sequences.

Kalata B1 has a lovely backstory worth knowing: it was first identified from a plant used in traditional African medicine, where a tea made from it was used to aid childbirth. Researchers investigating why the tea worked found this remarkably stable peptide.

Grafting: using the knot as a delivery van

Here is why drug designers are excited. If the knot provides stability and the loops can vary, you can replace a loop with a different short sequence, one that has a useful biological activity. The knot then protects that sequence.

The chapter describes exactly this. Researchers grafted fragments from the proteins laminin, osteopontin and VEGF (a growth factor that promotes blood vessel growth) onto the MCoTI-II cyclotide and onto another small cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1). The grafted versions were more stable than the original linear sequences and showed blood vessel growth activity in living animals at nanomolar concentrations.

In effect, the cyclotide becomes a protective vehicle for a short active message that would otherwise be destroyed.

Venoms, rebuilt as rings

The chapter describes a related approach: taking natural peptides that are linear but full of disulfides, and joining their ends to make them more stable.

Conotoxin Vc1.1 comes from the venom of cone snails. It is 16 amino acids long with two disulfide bridges, and acts on receptors in the nervous system, including GABA-B receptors, which is why it has been studied as a potential pain treatment. The chapter reports that a backbone-cyclised version was more stable than the linear form and more potent at GABA-B receptors.

Chlorotoxin comes from scorpion venom. It is 36 amino acids with four disulfide bridges. The chapter notes something remarkable about it: it binds to glioma cells, a type of brain tumour, but not to normal cells. That selectivity has made it a basis for imaging agents that help surgeons distinguish tumour from healthy tissue, and for nanoparticles and targeted gene therapy tools. Cyclising it improved its stability too.

How they are made

The chapter describes two synthesis routes. The group's preferred method builds the linear chain with a thioester at one end and a cysteine at the other, then joins the ends using the native chemical ligation reaction from Part 1 of this series, but within a single molecule, closing the ring. They prefer Boc chemistry for this, despite the need for hydrogen fluoride, because of its higher yields.

Folding the three bridges into the right knot, the chapter admits, tends to be done by trial and error for each peptide. But they note that for knotted peptides, cyclising and folding together often gives the natural arrangement in high yield without forcing it.

Why this matters to you

Cyclotides and cyclised venom peptides point at the future of peptide medicine. One of the biggest limitations of peptide drugs has been their fragility: short half-lives and poor oral absorption. Nature's knotted rings show that peptides can be made robust enough to survive conditions that would destroy ordinary ones.

It is also a reminder of where many peptide medicines come from: plants, snails, scorpions, lizards. The GLP-1 drug exenatide, for example, was developed from a peptide in Gila monster venom. Some of the most useful molecules in medicine started out as a creature's way of defending itself.

This series explains laboratory chemistry for general readers. It is not a guide to making or using any compound. Nothing here is medical advice.