Evaluation of the Spider ( Phlogiellus genus) Phlotoxin 1 and Synthetic Variants as Antinociceptive Drug Candidates

• Published in: Toxins Vol. 11; no. 9; pp. 484 - 18
• Main Authors: Gonçalves, Tânia C, Lesport, Pierre, Kuylle, Sarah, Stura, Enrico, Ciolek, Justyna, Mourier, Gilles, Servent, Denis, Bourinet, Emmanuel, Benoit, Evelyne, Gilles, Nicolas
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 22.08.2019; MDPI
• Subjects: Affinity; Alanine; Amino acids; Analgesics; Automation; Calcium; Calcium channels (voltage-gated); Cell lines; Chronic pain; Computer applications; Disulfide bonds; Drug development; Electrophysiology; FDA approval; Homology; human voltage-gated ion channel subtypes; Ion channels; Life Sciences; Liquid chromatography; Molecular structure; mouse model of NaV1.7-mediated pain; Narcotics; NaV1.7 channel subtype; Neurons and Cognition; Opioids; Pain; Pain perception; Peptides; Phlogiellus spider; phlotoxin 1; Potassium; Potassium channels (voltage-gated); Selectivity; Sodium channels (voltage-gated); Target recognition; Toxins; Venom; Venom toxins; mouse model of NaV1.7-mediated pain; human voltage-gated ion channel subtypes; NaV1.7 channel subtype; Phlogiellus spider; phlotoxin 1
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins11090484

Over the two last decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. At present, approximately 20 potential analgesic toxins, mainly from spider venoms, are known to inhibit with high affinity the Na 1.7 subtype of voltage-gated sodium (Na ) channels, the most promising genetically validated antinociceptive target identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) Na 1.7 subtype. The synthetic homologue of PhlTx1 was generated and equilibrated between two conformers on reverse-phase liquid chromatography and exhibited potent analgesic effects in a mouse model of Na 1.7-mediated pain. The effects of PhlTx1 and 8 successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNa subtypes, as well as two cardiac targets, the hCa 1.2 and hK 11.1 subtypes of voltage-gated calcium (Ca ) and potassium (K ) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNa 1.1-1.3 and 1.5-1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNa 1.4 and 1.8, hCa 1.2 and hK 11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of Na 1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the Na 1.7 subtype was at least five times higher than that of the wild-type peptide. Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure-activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the Na 1.7 subtype, but without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates.