Knottins provide useful scaffolds or leads for drug design

  • ‣ Knottins are exceptional in that they are very small proteins yet with particularly well-defined scaffolds and remarkably high stability.
  • ‣ Also remarkable is the fact that knottins with very similar 3D structures have virtually no sequence identity except for cysteines. This observation has led to the conclusion that the knottin scaffold is very sequence tolerant.
  • ‣ These remarkable features suggest that knottins can provide excellent lead molecules or elementary scaffold in drug design studies [Chiche et al., 2004; Craik et al., 2006; Werle et al., 2006; Moore et al., 2012], and in biotechnological applications [Cox et al., 2016; Moore et al., 2013; Glotzbach et al., 2013; Barba et al., 2012].
  • ‣ Main efforts along this way are outlined below.
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Cell internalization

Circular permutations

Computer simulations

Homology modeling



Protein engineering

Protein engineering

Knottin peptide-drug conjugate

Knottin peptide-drug conjugate (KDC) are a promising tools to selectively deliver drug to a target in vivo [Currier et al., 2016; Cox et al., 2016; Kim et al., 2015].

An engineered KDC to gemcitabine is able to binds to tumor cells with high affinity using integrin, penetrate cell, delivers drugs intracellularly, and is a highly potent inhibitor of various malignant cells [Cox et al., 2016; Kim et al., 2015].

Optical imaging of tumor using engineered knottin

Tumor cell can be targeted and illuminated with a fluorescent, engineered knottin, that binds with high affinity to integrin [Moore et al., 2013a; Moore et al., 2013b; Zhu et al., 2014].

Engineering and optimization of high-affinity inhibitors of protease

Yeast surface-displayed libraries of knottin were produced and screened for a matriptase-1 inhibitors activity. A subnanomolar inhibitor was discovered [Glotzbach et al., 2013].

Redox-Active Metallopeptides based on knottin scaffold

Cupricyclin-1 and -2, two novel enginnered knottin able to bind one Cu2+ ion with nanomolar affinity have been designed. These copper-based biocatalysts display redox activity and catalyze the dismutation of superoxide anions [Barba et al., 2012].

Knottins with new molecular recognition characteristics

An interesting review on using knottin for binding specific molecular targets [Moore et al., 2012].

Cyclooctapeptides as constrained analogues of the neurotoxin omega-agatoxine IVB

Constrained analogs of the binding loop of omega-agatoxine IVB were obtained via ring cyclization of octapeptides by side chain-side chain lactonization [Minta et al., 2008].

Polytides (Polymer-Peptide hybrids) as a tool to improve bioavailability of small disulfide-rich proteins

Replacement of nonessential amino acid residues with isostere polymeric spacers in conotoxin SIIIA resulted in more potent and longer-lasting analgesics when compared to SIIIA. It is proposed that polytides provide a promising strategy for transforming disulfide-rich peptides into therapeutics [Green et al., 2007].

Grafting active peptides into EETI-II and the minimized Agouti-related protein

Thrombopoietin is the primary regulator of platelet production. Peptides shown to act as high-affinity thrombopoietin antagonists were grafted into the active loops of EETI-II (loop 'a') and of the minimized Agouti-related protein AGRP(87-132) , loop 'e'. Most interestingly, covalent dimerization of the engineered miniproteins through lysine cross-linking yielded potent bivalent agonists of the thrombopoietin receptor (c-Mpl) [Krause et al., 2007].
The RGD sequence (IPRGDYR) was also grafted into loop 'a' of EETI-II and both RGD and KGD sequences (RVAKGDWNDDT) were grafted into loop 'e' of AGRP(87-132) [Reiss et al., 2006]. The RGD-grafted EETI-II and AGRP were more potent when compared to the unsupported peptides. In contrast the KGD-grafted AGRP resulted in very poor platelet inhibition.

Structure-activity studies on the Agouti-Related Protein (AGRP)

The Agouti-Related Protein (AGRP) is an endogeneous antagonist of melanocortin receptors. It has been shown that the C-terminal knottin domain of AGRP, AGRP(87-120) known as the mini-AGRP, is sufficient to maintain biological activity [Jackson et al., 2002]. Interestingly, the shorter AGRP(91-122) containing only the two-disulfide Cystine-Stabilized β-sheet motif of the knottin fold, also displayed antagonist activity although with reduced potency [Wilczynski et al., 2005]. Moreover, chimeric peptides based on the mini-AGRP template, and obtained by grafting agonist peptides resulted in potent agonists of melanocorin receptors 1 and 3-5, however receptor selectivity was affected [Jackson et al., 2005].

Replacement of the inhibitory loop of EETI-II with a different inhibitory peptide

A seven-residue peptide, derived from the inhibitory loop of the the third domain of ovomucoid inhibitor from turkey, has been grafted onto EETI-II. The resulting peptide has been shown to inhibit porcine pancreatic elastase [Hilpert et al., 2002].

Role of the β-hairpin in omega-Atracotoxin-Hv1a

Omega-Atracotoxin-Hv1a is an insect-specific neurotoxin. Removal of the C-terminal hairpin has been shown to suppress the insecticidal activity, although the knottin fold remains essentially unaffected (compare 1AXH and 1HVW). Alanine scanning on the hairpin then indicated that spatially close residues Asn(27) and Arg(35) are essential for the activity [Tedford et al., 2001].

Replacement of the GPNG β-turn of Min-23 with the RT-loop of SH3 domains

The Nef protein of HIV binds to SH3 domains of Src protein kinases. As a first step toward the development of new anti-HIV drugs, chimeric peptides were built by grafting the RT-loop of Src protein kinases onto the Min-23 peptide [Heitz et al., 2000].

Chimeric analogs of omega-conotoxin MVIIA and MVIIC

MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. Chimeric analogs of MVIIA and MVIIC in which the N- and C-terminal halves were exchanged showed that residues important for the recognition of N-type channels are located in the N-terminal half, especially position 11, whereas essential residues for P/Q-type channel recognition are spread over the whole molecule. [Sato et al., 1997; Sato et al., 2000].

Circularization of conotoxins

MVIIA conotoxin has revealed interesting therapeutic potential. Chimeric molecules combining the essential residues of the MVIIA with the stable circular scaffold of Kalata B1 have been synthesized and could improve the bioavailability of the compound for drug usage [Craik et al., 2000].

Possible use of AAI as insecticid

The Knottin AAI (α-amylase inhibitor from Amaranth) is considered as a very promising head for development of insecticids against insects like Tenebrio molitor, which are responsible for huge harvest losses [Pereira et al., 1999].

Replacement of the inhibitory loop of EETI-II with two different epitopes

Replacing the inhibitory loop of EETI-II by a 13-residue epitope of Sendai virus L-protein or by a 17-residue epitope from human bone Gla-protein, resulted in correctly folded variants. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold [Christmann et al., 1999]

The chimeric Trypsin Carboxypeptidase Inhibitor

Early studies showed that grafting the C-terminal active site of the Knottin PCI (Potato Carboxypeptidase A Inhibitor) at the C-terminus of another Knottin EETI-II (Ecballium Elaterium Trypsin Inhibitor) does not affect the Knottin fold of EETI-II. The resulting chimeric molecule is able to inhibit carboxypeptidase A and trypsin either separately or simultaneously [Le-Nguyen et al, 1990; Chiche et al., 1993].