APETx4, a novel sea anemone toxin and a modulator of the cancer-relevant potassium channel KV10.1

[en] The human ether-à-go-go channel (hEag1 or KV10.1) is a cancer-relevant voltage-gated potassium channel that is overexpressed in a majority of human tumors. Peptides that are able to selectively inhibit this channel can be lead compounds in the search for new anticancer drugs. Here, we report the activity-guided purification and electrophysiological characterization of a novel KV10.1 inhibitor from the sea anemone Anthopleura elegantissima. Purified sea anemone fractions were screened for inhibitory activity on KV10.1 by measuring whole-cell currents as expressed in Xenopus laevis oocytes using the two-microelectrode voltage clamp technique. Fractions that showed activity on Kv10.1 were further purified by RP-HPLC. The amino acid sequence of the peptide was determined by a combination of MALDI- LIFT-TOF/TOF MS/MS and CID-ESI-FT-ICR MS/MS and showed a high similarity with APETx1 and APETx3 and was therefore named APETx4. Subsequently, the peptide was electrophysiologically characterized on KV10.1. The selectivity of the toxin was investigated on an array of voltage-gated ion channels, including the cardiac human ether-à-go-go-related gene potassium channel (hERG or Kv11.1). The toxin inhibits KV10.1 with an IC50 value of 1.1 µM. In the presence of a similar toxin concentration, a shift of the activation curve towards more positive potentials was observed. Similar to the effect of the gating modifier toxin APETx1 on hERG, the inhibition of Kv10.1 by the isolated toxin is reduced at more positive voltages and the peptide seems to keep the channel in a closed state. Although the peptide also induces inhibitory effects on other KV and NaV channels, it exhibits no significant effect on hERG. Moreover, APETx4 induces a concentration-dependent cytotoxic and proapoptotic effect in various cancerous and noncancerous cell lines. This newly identified KV10.1 inhibitor can be used as a tool to further characterize the oncogenic channel KV10.1 or as a scaffold for the design and synthesis of more potent and safer anticancer drugs. © 2017 by the authors. Licensee MDPI.

Disciplines :

Chemistry

Author, co-author :

Moreels, L.; Toxicology and Pharmacology, KU Leuven, Leuven, Belgium

Peigneur, S.; Toxicology and Pharmacology, KU Leuven, Leuven, Belgium

Galan, D. T.; Toxicology and Pharmacology, KU Leuven, Leuven, Belgium

De Pauw, Edwin ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)

Béress, L.; Immunology and Rheumatology, Section of Peptide Chemistry, Hannover Medical School (MHH), Hannover, Germany

Waelkens, E.; Laboratory of Protein Phosphorylation and Proteomics, KU Leuven, Leuven, Belgium

Pardo, L. A.; Oncophysiology Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany

Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique

Tytgat, Jan; Toxicology and Pharmacology, KU Leuven, Leuven, Belgium

Language :

English

Title :

APETx4, a novel sea anemone toxin and a modulator of the cancer-relevant potassium channel KV10.1

Publication date :

13 September 2017

Journal title :

Marine Drugs

ISSN :

1660-3397

Publisher :

MDPI AG

Volume :

Issue :

Pages :

287

Peer reviewed :

Peer Reviewed verified by ORBi

Funders :

IWT - Agentschap voor Innovatie door Wetenschap en Technologie [BE]

Available on ORBi :

since 31 January 2018

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Bibliography

Torre, L.A.; Bray, F.; Siegel, R.L.; Ferlay, J.; Lortet-tieulent, J.; Jemal, A. Global Cancer Statistics, 2012. CA Cancer J. Clin. 2015, 65, 87–108.
World Health Organization (WHO). Cancer: Fact Sheet. February 2017. Available online: http://www.who.int/mediacentre/factsheets/fs297/en/ (accessed on 14 June 2017).
American Cancer Society Global Cancer Facts & Figures 3rd Edition. Am. Cancer Soc. 2015.
Mello de Queiroz, F.; Suarez-Kurtz, G.; Stühmer, W.; Pardo, L.A. Ether à go-go potassium channel expression in soft tissue sarcoma patients. Mol. Cancer 2006, 5, 42.
Agarwal, J.R.; Griesinger, F.; Stühmer, W.; Pardo, L.A. The potassium channel Ether à go-go is a novel prognostic factor with functional relevance in acute myeloid leukemia. Mol. Cancer 2010, 9, 18.
Hemmerlein, B.; Weseloh, R.M.; Mello de Queiroz, F.; Knötgen, H.; Sánchez, A.; Rubio, M.E.; Martin, S.; Schliephacke, T.; Jenke, M.; Stümer, W.; et al. Overexpression of Eag1 potassium channels in clinical tumours. Mol. Cancer 2006, 5, 41.
Asher, V.; Khan, R.; Warren, A.; Shaw, R.; Schalkwyk, G.V.; Bali, A.; Sowter, H.M. The Eag potassium channel as a new prognostic marker in ovarian cancer. Diagn. Pathol. 2010, 5, 78.
Ding, X.; Luo, H.; Jin, X.; Yan, J.; Ai, Y. Aberrant expression of Eag1 potassium channels in gastric cancer patients and cell lines. Med. Oncol. 2007, 24, 345–350.
Ding, X.-W.; Wang, X.-G.; Luo, H.-S.; Tan, S.-Y.; Gao, S.; Luo, B.; Jiang, H. Expression and prognostic roles of Eag1 in resected esophageal squamous cell carcinomas. Dig. Dis. Sci. 2008, 53, 2039–2044.
Ding, X.-W.; Yan, J.-J.; An, P.; L, P.; Luo, H.-S. Aberrant expression of ether à go-go potassium channel in colorectal cancer patients and cell lines. World J. Gastroenterol. 2007, 13, 1257–1261.
Martin, S.; Lino de Oliveira, C.; Mello de Queiroz, F.; Pardo, L.A.; Stühmer, W.; Del Bel, E. Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain. Neuroscience 2008, 155, 833–844.
Saganich, M.J.; Machado, E.; Rudy, B. Differential expression of genes encoding subthreshold-operating voltage-gated K+ channels in brain. J. Neurosci. 2001, 21, 4609–4624.
Pardo, L.A.; Sühmer, W. Eag1 as a cancer target. Expert Opin. Ther. Targets 2008, 12, 837–843.
Pardo, L.A.; del Camino, D.; Sánchez, A.; Alves, F.; Brüggemann, A.; Beckh, S.; Stühmer, W. Oncogenic potential of EAG K(+) channels. EMBO J. 1999, 18, 5540–5547.
Weber, C.; Mello de Queiroz, F.; Downie, B.R.; Suckow, A.; Stühmer, W.; Pardo, L.A. Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference. J. Biol. Chem. 2006, 281, 13030–13037.
García-Ferreiro, R.E.; Kerschensteiner, D.; Major, F.; Monje, F.; Stühmer, W.; Pardo, L.A. Mechanism of block of hEag1 K+ channels by imipramine and astemizole. J. Gen. Physiol. 2004, 124, 301–317.
Gomez-Lagunas, F.; Carrillo, E.; Pardo, L.A.; Stühmer, W. Gating Modulation of the Tumor-Related Kv10.1 Channel by Mibefradil. J. Cell. Physiol. 2016.
Garg, V.; Stary-Weinzinger, A.; Sanguinetti, M.C. ICA-105574 interacts with a common binding site to elicit opposite effects on inactivation gating of EAG and ERG potassium channels. Mol. Pharmacol. 2013, 83, 805–813.
Gómez-Varela, D.; Zwick-Wallasch, E.; Knötgen, H.; Sánchez, A.; Hettmann, T.; Ossipov, D.; Weseloh, R.; Contreras-Jurado, C.; Rothe, M.; Stühmer, W.; et al. Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity. Cancer Res. 2007, 67, 7343–7349.
Hartung, F.; Stühmer, W.; Pardo, L.A. Tumor cell-selective apoptosis induction through targeting of K(V)10.1 via bifunctional TRAIL antibody. Mol. Cancer 2011, 10, 109.
Wulff, H.; Castle, N.A.; Pardo, L.A. Voltage-gated potassium channels as therapeutic targets. Nat. Rev. Drug Discov. 2009, 8, 982–1001.
Pardo, L.A.; Gómez-Varela, D.; Major, F.; Sansuk, K.; Leurs, R.; Downie, B.R.; Tietze, L.F.; Stuhmer, W. Approaches targeting Kv10.1 open a novel window for cancer diagnosis and therapy. Curr. Med. Chem. 2012, 19, 675–682.
Lewis, R.J.; Garcia, M.L. Therapeutic potential of venom peptides. Nat. Rev. Drug Discov. 2003, 2, 790–802.
Moreels, L.; Peigneur, S.; Yamaguchi, Y.; Vriens, K.; Waelkens, E.; Zhu, S.; Thevissen, K.; Cammue, B.P.A.; Sato, K.; Tytgat, J. Expanding the pharmacological profile of κ-hefutoxin 1 and analogues: A focus on the inhibitory effect on the oncogenic channel Kv10.1. Peptides 2016.
Srinivasan, K.N.; Sivaraja, V.; Huys, I.; Sasaki, T.; Cheng, B.; Kumar, T.K.S.; Sato, K.; Tytgat, J.; Yu, C.; San, B.C.C.; et al. kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity. J. Biol. Chem. 2002, 277, 30040–30047.
Schumacher, M.; Kelkel, M.; Dicato, M.; Diederich, M. Gold from the sea: Marine compounds as inhibitors of the hallmarks of cancer. Biotechnol. Adv. 2011, 29, 531–547.
Kiuru, P.; D’Auria, M.V.; D.Muller, C.; Tammela, P.; Vuorela, H.; Yli-Kauhaluoma, J. Exploring Marine Resources for Bioactive Compounds. Planta Med. 2014, 80, 1234–1246.
Zheng, L.-H.; Wang, Y.-J.; Sheng, J.; Wang, F.; Zheng, Y.; Lin, X.-K.; Sun, M. Antitumor Peptides from Marine Organisms. Mar. Drugs 2011, 9, 1840–1859.
Suarez-Jimenez, G.-M.; Burgos-Hernandez, A.; Ezquerra-Brauer, J.-M. Bioactive Peptides and Depsipeptides with Anticancer Potential: Sources from Marine Animals. Mar. Drugs 2012, 10, 963–986.
Dyshlovoy, S.; Honecker, F. Marine Compounds and Cancer: Where Do We Stand? Mar. Drugs 2015, 13, 5657–5665.
Kim, S.; Kalimuthu, S. Introduction to Anticancer Drugs from Marine Origin. In Handbook of Anticancer Drugs from Marine Origin; Springer: Berlin/Heidelberg, Germany, 2014; pp. 1–13, ISBN 9783319071459.
Rocha, J.; Peixe, L.; Gomes, N.C.M.; Calado, R. Cnidarians as a source of new marine bioactive compounds—An overview of the last decade and future steps for bioprospecting. Mar. Drugs 2011, 9, 1860–1886.
Frazão, B.; Vasconcelos, V.; Antunes, A. Sea anemone (cnidaria, anthozoa, actiniaria) toxins: An overview. Mar. Drugs 2012, 10, 1812–1851.
Pennington, M.W.; Chang, S.C.; Chauhan, S.; Huq, R.; Tajhya, R.B.; Chhabra, S.; Norton, R.S.; Beeton, C. Development of Highly Selective Kv1.3-Blocking Peptides Based on the Sea Anemone Peptide ShK. Mar. Drugs 2015, 13, 529–542.
A 4 Week Study of the Safety, Tolerability, and Pharmacodynamics of ShK-186 (Dalazatide) in Active Plaque Psoriasis. 2015. Available online: http://clinicaltrials.gov (accessed on 12 September 2017).
Multiple Ascending Dose Safety Study of ShK-186 (Dalazatide) in Healthy Volunteers (2015). Available online: http://clinicaltrials.gov (accessed on 12 September 2017).
Bruhn, T.; Schaller, C.; Schulze, C.; Sanchez-Rodriguez, J.; Dannmeier, C.; Ravens, U.; Heubach, J.F.; Eckhardt, K.; Schmidtmayer, J.; Schmidt, H.; Aneiros, a.; Wachter, E.; Béress, L. Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon 2001, 39, 693–702.
McCommas, S.A. Relationships within the family Actiniidae (Cnidaria, Actiniaria) based on molecular characters. Hydrobiologia 1991, 216–217, 509–512.
King, G.F.; Gentz, M.C.; Escoubas, P.; Nicholson, G.M. A rational nomenclature for naming peptide toxins from spiders and other venomous animals. Toxicon 2008, 52, 264-276.
Oliveira, J.S.; Fuentes-Silva, D.; King, G.F. Development of a rational nomenclature for naming peptide and protein toxins from sea anemones. Toxicon 2012, 60, 539-550.
Peigneur, S.; Béress, L.; Möller, C. A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins. FASEB J. 2012, 26, 5141-5151.
Diochot, S.; Loret, E.; Bruhn, T.; Béress, L.; Lazdunski, M. APETx1, a new toxin from the sea anemone Anthopleura elegantissima, blocks voltage-gated human ether-a-go-go-related gene potassium channels. Mol. Pharmacol. 2003, 64, 59-69.
Wang, J.; Salata, J.J.; Bennett, P.B. Saxitoxin is a gating modifier of HERG K+ channels. J. Gen. Physiol. 2003, 121, 583-598.
Zhang, M.; Liu, X.-S.X.; Diochot, S.; Lazdunski, M.; Tseng, G.-N.N. APETx1 from sea anemone Anthopleura elegantissima is a gating modifier peptide toxin of the human ether-a-go-go-related potassium channel. Mol. Pharmacol. 2007, 72, 259-268.
Catterall, W.A.; Cestèle, S.; Yarov-Yarovoy, V.; Yu, F.H.; Konoki, K.; Scheuer, T. Voltage-gated ion channels and gating modifier toxins. Toxicon 2007, 49, 124-141.
Swartz, K.J.; MacKinnon, R. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K+ channels. Neuron 1997, 18, 675-682.
Takahashi, H.; Kim, J., II; Min, H.J.; Sato, K.; Swartz, K.J.; Shimada, I. Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins. J. Mol. Biol. 2000, 297, 771-780.
Wang, J.M.; Roh, S.H.; Kim, S.; Lee, C.W.; Kim, J., II; Swartz, K.J. Molecular surface of tarantula toxins interacting with voltage sensors in K(v) channels. J. Gen. Physiol. 2004, 123, 455-467.
Sanguinetti, M.C.; Tristani-Firouzi, M. hERG potassium channels and cardiac arrhythmia. Nature 2006, 440, 463-469.
Shao, X.-D.; Wu, K.-C.; Guo, X.-Z.; Xie, M.-J.; Zhang, J.; Fan, D.-M. Expression and significance of HERG protein in gastric cancer. Cancer Biol. Ther. 2008, 7, 45-50.
Asher, V.; Warren, A.; Shaw, R.; Sowter, H.; Bali, A.; Khan, R. The role of Eag and HERG channels in cell proliferation and apoptotic cell death in SK-OV-3 ovarian cancer cell line. Cancer Cell Int. 2011, 11, 6.
Asher, V.; Sowter, H.; Shaw, R.; Bali, A.; Khan, R. Eag and HERG potassium channels as novel therapeutic targets in cancer. World J. Surg. Oncol. 2010, 8, 113.
Diochot, S.; Baron, A.; Rash, L.D.; Deval, E.; Escoubas, P.; Scarzello, S.; Salinas, M.; Lazdunski, M. A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons. EMBO J. 2004, 23, 1516-1525.
Jensen, J.E.; Cristofori-Armstrong, B.; Anangi, R.; Rosengren, K.J.; Lau, C.H.Y.; Mobli, M.; Brust, A.; Alewood, P.F.; King, G.F.; Rash, L.D. Understanding the molecular basis of toxin promiscuity: The analgesic sea anemone peptide APETx2 interacts with acid-sensing ion channel 3 and hERG channels via overlapping pharmacophores. J. Med. Chem. 2014, 57, 9195-9203.
Diana Urrego; Naira Movsisyan; Roser Ufartes; Luis A. Pardo. Periodic expression of Kv10.1 contributes to G2/M progression of cancer and non- transformed cells. Cell Cycle 2016, 15, 99-811.
Kourie, J.I.; Shorthouse, A.A. Properties of cytotoxic peptide-formed ion channels. Am. J. Physiol. Cell Physiol. 2000, 278, C1063-C1087.
Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera-A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605-1612.
Webb, B.; Sali, A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 2016, 54, 5.6.1–5.6.37.
Goujon, M.; Mcwilliam, H.; Li, W.; Valentin, F.; Squizzato, S.; Paern, J.; Lopez, R. A new bioinformatics analysis tools framework at EMBL–EBI. Nucleic Acids Res. 2010, 38, W695–W699.