Chemical synthesis, backbone cyclization and oxidative folding of cystine-knot peptides: promising scaffolds for applications in drug design

Abstract

Cystine-knot peptides display exceptional structural, thermal, and biological stability. Their eponymous motif consists of six cysteine residues that form three disulfide bonds, resulting in a notably rigid structural core. Since they highly tolerate either rational or combinatorial changes in their primary structure, cystine knots are considered to be promising frameworks for the development of peptide-based pharmaceuticals. Despite their relatively small size (two to three dozens amino acid residues), the chemical synthesis route is challenging since it involves critical steps such as head-to-tail cyclization and oxidative folding towards the respective bioactive isomer. Herein we describe the topology of cystine-knot peptides, their synthetic availability and briefly discuss potential applications of engineered variants in diagnostics and therapy.

Figure 1

Cartoon diagrams of prototypical cystine knots. Loops are depicted in light blue and numbered according to their appearance in the sequence, α-helices in dark blue, β-sheets in red, and cysteines in yellow with Roman numerals according to their appearance in the sequence. (a) Möbius cyclotide kalata B1. (PDB-ID: 1NB1) (b) Bracelet cyclotide cycloviolacin O2. (PDB-ID: 2KNM) (c) Acyclic inhibitor cystine knot ocMCoTI-II (PDB-ID: 2IT8). Structures modeled with Yasara Ver. 12.4.1.

Figure 2

Sequence alignment of certain cystine knots. Cystine connections as well as head-to-tail macrocyclization motif are indicated.

Figure 3

Common strategies for backbone cyclization. Biosynthetic methods are depicted in green, chemical methods in blue, hybrid strategies are shown in turquoise. References: Intein cyclization: [39,40,41]; hydrazone cyclization: [43]; proteolytic cyclization: [44,45]; thia-zip cyclization: [46,47].

Figure 4

In vitro folding pathways of cystine-knot peptides from different families. Most ICK peptides are thought to proceed to the folded form via formation of two-disulfide native-like intermediate. Folding of CCK peptides may either follow a similar path or proceed via a non-native 3-cystine intermediate. Structures were modeled and energy-minimized with Yasara Ver. 12.4.1.

Figure 5

Proposed cyclization via native chemical ligation.