Knottins display a variety of biological functions


  • ‣ Some knottins are thought to perform their action via interaction with a receptor.
  • ‣ This has led several authors to use the slightly restrictive name "Inhibitor Cystine Knot" (ICK)
  • ‣ Different activities are located on different parts of the molecules, and almost all Knottin loops have been implied in one ore more function.
  • ‣ The major known biological functions exerted by Knottins are briefly outlined below.
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Analgesics

Anthelmintic

Anti-ED

Antimalarial

Antimicrobials

Antitumors

Protease inhibitors

Toxins

Insecticids

Others

Analgesic action

Conotoxins and spider toxins that block voltage-sensitive calcium chanels are potent analgesics

  • Neurotoxin Tx3-6 from brazilian armed spider displayed higher efficacy and long-lasting analgesia in a thermal model of pain, when compared with omega-conotoxin MVIIA. Tx3-6 was also more effective to reverse previously installed persistent chemical and neuropathic pain. It is suggested that Tx3-6 might be useful in the control of persistent pathological pain [Souza et al., 2008].
  • The tarantula spider toxin GsMTx4 was shown to reduce mechanical and neuropathic pain . However, GsMTx4 did not affect response to thermal stimuli [Park et al., 2008].
  • Omega-conotoxin MVIIA (Ziconotide) is a potent analgesic and has been approved for the treatment of severe chronic pain [Wallace 2006].
  • Anthelmintic activity

    Cyclotides possess anthelmintic activity

    • Several Kalata and cycloviolacin peptides were shown to display significant activity in inhibiting larvae development and adult motility of Haemonchus contortus and Trichostrongylus colubriformis larvae, two important gastrointestinal nematode parasites of sheep. The activity displays no chiral selectivity and appears to be related to clustered basic residues suggesting that membrane interaction rather than interaction with a receptor is involved in the cyclotide anthelmintic activity [Colgrave et al., 2008].

    Anti Erectile Dysfunction (ED)

    A spider toxin induces penile erection in rats

    • The spider toxin Tx2-6 was shown to induce and facilitate the ganglionic-stimulated penile erection in rats. It also displayed a potentiation effect on penile erection of animals presenting severe erectile dysfunction such as the deoxycorticosterone-acetate-salt hypertensive rats. The potentiating effect of Tx2-6 on erection seems to be mediated by relaxation of the vasculature and smooth muscle in the corpus cavernosum induced by the release of NO [Nunes et al., 2008].
    • Also note that the agouti-related and the agouti-signaling proteins act on the melanocortin system that is involved in sexual function.

    Antimalarial activity

    Toxins from the venom of the tarantula Psalmopoeus cambridgei display strong antiplasmodial activity

    Antimicrobials

    Several knottins display antimicrobial activities

    • A review about antimicrobial activity of plant peptides, including some knottins [ Tam et al., 2015 ]
    • In Bemisia tabaci, three knottin-like antimicrobial peptide genes are activated in response to the pathogen Pseudomonas aeruginosa [ Zhang et al., 2014 ]
     
     Source  Knottin  PDB ID  Inhibits growth of
           gram +  gram -  fungi
     Plant  PAFPS  1DKC    

    +
     Plant  Kalata B1   1KAL 1K48 1NB1

    +
       
     Plant  Circulin A  1BH4

    +
       
     Plant  Circulin B  

    +

    +
     
     Plant  Cyclopsychotride  

    +

    +

    +
               
     Crab  Tachystatin  1CIX

    +

    +

    +

    Antitumors

    Antitumor activities have been shown for PCI and cyclotides

    • The Potato Carboxypeptidase inhibitor PCI is an antagonist of human EGF and inhibits tumor cell growth [Blanco-Aparicio et al., 1998 ]
    • The cyclotides varv A, varv F, and cycloviolacin O2 display cytotoxicity for several human tumor cell lines [Lindholm et al., 2002]. The activity profiles differed significantly from those of antitumor drugs in clinical use, suggesting a new mode of action for these cyclotides.
    • Cycloviolacin O2 Disrupts Lipid Membranes. Cytotoxic, antimicrobial, hemolytic, and insecticidal effects are likely all connected to membrane disruption. [Svangard et al., 2007 ].

    Protease inhibitors

    The first known Knottins were protease inhibitors

    Inhibitors with the knottin scaffold have been isolated from plants. Insects and phytopathogenic microorganisms secrete enzymes causing proteolytic digestion or proteins. It is likely that, to defend themselves from these attacks, plants have developped expression of proteinase inhibitors. [Habib & Fazili, 2007]
    • Cyclotide psysol 2: inhibitor of human prolyl oligopeptidase (POP) [Hellinger et al., 2015].
    • Roseltide: a knottin-type neutrophil elastase inhibitor derived from Hibiscus sabdariffa [Loo et al., 2016].
    • PCI: The potato carboxypeptidase A inhibitor PCI inhibits its cognate enzyme through its C-terminus [Rees & Lipscomb, 1982].
    • Squash: The serine protease inhibitors of the "squash" family inhibit their cognate enzyme through a canonical loop located between Cys1 and Cys2 [Bode et al., 1989].
    • α-Amylase: The α-amylase inhibitor from Amaranth AAI, inhibits α-amylase from insects using a new mode of action. The interaction surface includes two segments: (i) the loop between Cys1 and Cys2 and (ii) the last strand of the β-hairpin [Pereira et al., 1999].

    Toxins

    Toxic knottins inhibit ion channels, but the mechanism of inhibition remains unknown.

    It has been shown that the spider toxin VSTX1 reaches its target by partitioning into the lipid membrane [Lee & MacKinnon, 2004].

    Moreover, both the spider toxin GsMTx4, and its enantiomer, enGsMTx4, were shown to modify the gating of the target ion channel, ruling out the traditional lock-and-key model of ligand-protein interactions. It is hypothesized that GsMTx4 and enGsMTx4 alter the lipid packing at the bilayer/solution/channel interface [Suchyna et al., 2004; Garcia, 2004]. Molecular dynamics simulations have evidenced two modes of binding between GsMTx4 and membranes. A deep binding is favored when a DPPC (dipalmitoyl-phosphatidylcholine) membrane is used at 310 K, indicating that toxin binding with biological membranes is affected by the structure of the lipid acyl chains [Nishizawa & Nishizawa, 2007].

    Further studies have shown however that bilayer partitioning is not a universal property of the toxic knottins that interact with ion channels [Posokhov et al., 2007].

    More recently, the asteropsin A, an unusual cystine-crosslinked peptide from porifera, has been shown to induce neuronal Ca2+ influx when it was administered together with the Na+ channel activator veratridine [Li et al., 2013].

    In 2016, a sodium channel inhibitor ISTX-I with a novel structure has been discovered and provides a new hint at the evolutionary link between two toxin folds [Rong et al., 2016].

    Toxic Knottins are able to block many ion channels

    exemples are shown below

     Channel  Knottin  PDB ID
     Sodium channel agonist/antagonist  delta-conotoxin TXVIA  1FU3
     mu1 sodium channel receptor  conotoxin EVIA  1G1Z
     Potassium channel blocker  kappa-conotoxin PVIIA  1AV3 1KCP
     kv4-2 Potassium channel blocker  Heteropodatoxin-2  1EMX
     Potassium channel gating modifier  Hanatoxin-1  1D1H
     Calcium channel blocker  omega-conotoxin GVIA
    omega-atracotoxin HV2A
     2CCO 1G9P
     N-type calcium channel blocker  omega-conotoxin MVIIA  1MVJ 1MVI 1DW4 1I26
     L-type calcium channel blocker  omega-conotoxin TXVII  1F3K
     Calcium channel gating modifier  omega-grammotoxin SIA  1KOZ
     Sodium channel inhibitor  Neurotoxin ISTX-I  2NDI
     Sodium channel inhibitor  Conotoxin mu O-GVIIJ  2N8H
         

    Insecticids

    Few Knottins have shown insecticidal activity

    • Orally active insecticidal peptide (OAIP-1) (PDB ID: 2LL1) from venom of the Australian tarantula Selenotypus plumipes [Hardy et al., 2013].
    • J-Atracotoxin-HV1C (PDB ID: 1DL0)
    • Kalata B1 and other cyclotides display insecticidal activities. It has been shown that the insecticidal and hemolytic activity of kalata B1 are both dependent on a cluster of hydrophilic residues separate from the membrane binding face. It is postulated that specific self-association, in combination with membrane binding mediates cyclotide bioactivities [Simonsen et al., 2008]. Kalata B1 has been shown to disrupt epithelial cells in the midgut of lepidopteran larvae and consequently to negatively affect nutrient uptake [Barbeta et al., 2008].
    • α-Amylase inhibitor AAI (PDB ID: 1CLV)
    • Plant proteinase inhibitors: Leaf-specific over-expression of the potato inhibitor PI-II and of the knottin PCI (carboxypeptidase inhibitor) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels of the transgenes [Abdeen et al., 2005].

    Others

    Besides above activities, it is worth noting that

    • Knottin-1 gene of whitefly [ Hariton Shalev et al., 2016] displays antiviral activity on tomato yellow leaf curl virus.

    • Circulin A, cycloviolins A-D and kalata B8 [ Hallock et al., 2000; Daly et al., 2006] displays anti-HIV activity.

    • Cyclopsychotride A inhibits neurotensin binding to HT-29 cell membranes.

    • Cycloviolacin H4 displays the highest hemolytic activity among cyclotides. It has been proposed that this activity correlates with the size of a surface-exposed hydrophobic patch [ Chen et al., 2006].

    • Kalata B1 is used in traditional medicine for its uterotonic activity

    • Agouti and Agouti-related proteins (AgRP) are endogeneous antagonists of the melanocortin receptors (MCRs) [Gantz and Fung, 2003; Stutz et al., 2005; Pritchard et al., 2005]. The normal role of agouti in mammals is to determine coat color in conjunction with MC1R and the melanocortin peptide α-melanocyte-stimulating hormone (α-MSH). Hence the term agouti that refers to a hair color pattern characterized by subapical yellow bands. Interestingly, agouti also acts as an antagonist of hypothalamic α-MSH, a major satiety factor, and is thus involved in obesity.
      Studies in humans revealed associations of AGRP single nucleotide polymorphisms with resistance to obesity. Administration or overexpression of AgRP increases intake of fat diet On the other hand, absence or reduced functionality of AgRP leads to an increased lifespan [Ilnytska & G. Argyropoulos, 2008; Tracy et al., 2008]. As the melanocortin system is involved in diverse physiological functions, including pigmentation, energy homeostasis, inflammation, fat intake and sexual function. Melanocortin-based drugs are therefore considered for the treatment of skin cancer, obesity, erectile dysfunction, inflammatory diseases, pain, etc.