Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering

Abstract

Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.

Centipede venoms as a source of drug leads

“…For example, no degradation of Ssm6a was observed when the peptide was incubated for one week in human plasma in vitro, and the peptide was shown to be highly resistant to thermal denaturation in 4 M urea; remarkably, the midpoint of the thermal unfolding transition (T m ) was even very high (71˚C) in 8 M urea [21]. Hence, the HAND peptide scaffold appears to be an ideal structural template for engineering therapeutically useful biological functions, as has been done successfully with inhibitor cystine knot toxins [40,41].…”

“…The internal cross-bracing provided by covalent disulfide bonds creates a rigid structural framework that provides resistance to chemical and thermal denaturation as well as degradation by proteases [4,21,22,34,35]. Although peptides of similar structure can harbor a diversity of interesting pharmacological properties, the characterization of new disulfide frameworks is of particular interest from a drug discovery perspective as they are more likely to exhibit novel pharmacology [36] and expand the repertoire of structural templates for bioengineering purposes [37][38][39][40][41].…”

“…Thus, the next decade promises to be an exciting period where fundamental studies of centipede venom begin to be translated into therapeutic applications. Moreover, it will be particularly interesting to see whether some of the unique disulfide-constrained peptide scaffolds present in centipede venoms, such as the HAND toxin fold [35,60], prove as useful as the ubiquitous inhibitor cystine knot fold for engineering desirable therapeutic and diagnostic functions [40,41,100]. …”

Overlooked Short Toxin-Like Proteins: A Shortcut to Drug Design

“…Based on the observation that many short animal toxins are rich in cysteines [ 49 , 50 ], we focused on a subset of short proteins (<75 amino acids) that can be used for discoveries towards peptide therapy [ 51 ]. The goal of our study is to present a systematic approach for identifying insects’ toxin-like proteins TOLIPs (iTOLIPs).…”

Targeting the tumor vasculature with engineered cystine-knot miniproteins

“…Their simple architecture allows convenient chemical manufacturing and site-directed conjugation of, e.g. fluorophores, chelators, or toxins 23,24 . The exposed surface loops of cystine-knot miniproteins tolerate sequence modifications and insertions, facilitating the generation of target-binding capabilities via loop grafting or directed evolution based protein design.…”