Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Mar Drugs
2012 Jul 01;107:1511-1527. doi: 10.3390/md10071511.
Show Gene links
Show Anatomy links
Cyclisation increases the stability of the sea anemone peptide APETx2 but decreases its activity at acid-sensing ion channel 3.
Jensen JE
,
Mobli M
,
Brust A
,
Alewood PF
,
King GF
,
Rash LD
.
???displayArticle.abstract???
APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.
Figure 1. Wild-type APETx2 and truncation analogues were made in a two-step approach whereby the two peptide chains were synthesised separately then joined via NCL. The cyclic analogues were synthesised as single peptide and cyclised using NCL.
Figure 2. (A) SOFAST-HMQC spectrum of APETx2_wt (black) overlaid with that of APETx2_1–39 (red). A crosspeak is present for each H-N bond (i.e., for each non-proline residue), including non-labile sidechain NH groups (Trp Nε-Hε, Asn Nδ-Hδ2, Gln Nε-Hε and Arg Nε-Hε). (B) Backbone 15N chemical shifts for APETx2_wt (black), APETx2_1–39 (red) and APETx2_3–42 (cyan).
Figure 3. (A) Overlaid HMQC spectra of APETx2_wt (black) and cAPETx2_6RL (green). (B) Overlay of the backbone 15N chemical shifts of APETx2_wt (black) and cyclic APETx2 with either a 6-residue (green), 7-residue (maroon), or 8‑residue (blue) linker.
Figure 4. Decay curves showing the stability of APETx2_wt (black), cAPETx2_6RL (green), cAPETx2_7RL (brown), and cAPETx2_8RL (blue) against proteolytic cleavage by (A) trypsin and (B) pepsin.
Figure 5. Comparison of the activity of wild-type APETx2 with cyclic and truncation analogues. Concentration-effect curves are shown for inhibition of rASIC3 currents by APETx2 wt (black), APETx2_1–39 (red) and APETx2_3–42 (cyan) (n = 6). The activity of the cyclic APETx2 analogues is shown only at 1 µM (n = 6).
Figure 6. (A) Surface representation of APETx2 highlighting residues potentially involved in its interaction with ASIC3: Cluster 1 (residues A3, S5, N8, K10, T39 and A41) is coloured red and Cluster 2 (residues Y16, R17, P18, R31 and T36) is coloured cyan. The N- and C-termini are coloured yellow and violet, respectively. Arg17 is coloured dark blue. (B) Surface representation of homology model of cAPETx2_6RL showing the six-residue linker in green. Potential residues interacting with ASIC3 are highlighted as for (A).
Adessi,
Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer's disease.
2003, Pubmed
Adessi,
Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer's disease.
2003,
Pubmed
Anangi,
Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3.
2010,
Pubmed
Blanchard,
Inhibition of voltage-gated Na(+) currents in sensory neurones by the sea anemone toxin APETx2.
2012,
Pubmed
,
Xenbase
Chagot,
Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels.
2005,
Pubmed
Clark,
Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII.
2005,
Pubmed
,
Xenbase
Clark,
The engineering of an orally active conotoxin for the treatment of neuropathic pain.
2010,
Pubmed
Deval,
ASIC3, a sensor of acidic and primary inflammatory pain.
2008,
Pubmed
Deval,
Acid-sensing ion channels in postoperative pain.
2011,
Pubmed
Diochot,
A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons.
2004,
Pubmed
,
Xenbase
Escoubas,
Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton-gated cation channels.
2003,
Pubmed
Fu,
Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study.
2002,
Pubmed
Gould,
Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery.
2011,
Pubmed
Halai,
Effects of cyclization on stability, structure, and activity of α-conotoxin RgIA at the α9α10 nicotinic acetylcholine receptor and GABA(B) receptor.
2011,
Pubmed
,
Xenbase
Jensen,
Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3.
2009,
Pubmed
,
Xenbase
Karczewski,
Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2.
2010,
Pubmed
King,
Structural basis for the topological specificity function of MinE.
2000,
Pubmed
King,
Venoms as a platform for human drugs: translating toxins into therapeutics.
2011,
Pubmed
Kwan,
Macromolecular NMR spectroscopy for the non-spectroscopist.
2011,
Pubmed
Lesner,
Sunflower trypsin inhibitor 1 as a molecular scaffold for drug discovery.
2011,
Pubmed
Lewis,
Therapeutic potential of venom peptides.
2003,
Pubmed
Li,
ASIC3 channels in multimodal sensory perception.
2011,
Pubmed
Linde,
Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides.
2008,
Pubmed
Lingueglia,
Acid-sensing ion channels in sensory perception.
2007,
Pubmed
Lovelace,
Cyclic MrIA: a stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter.
2006,
Pubmed
Norton,
Structures of sea anemone toxins.
2009,
Pubmed
Saez,
Spider-venom peptides as therapeutics.
2010,
Pubmed
Schanda,
Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds.
2005,
Pubmed
Schnölzer,
In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences.
1992,
Pubmed
Shiomi,
Novel peptide toxins recently isolated from sea anemones.
2009,
Pubmed
Sluka,
Acid-sensing ion channels: A new target for pain and CNS diseases.
2009,
Pubmed
Tugyi,
Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide.
2005,
Pubmed
Vetter,
Venomics: a new paradigm for natural products-based drug discovery.
2011,
Pubmed
Yamaguchi,
Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.
2010,
Pubmed