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

Abstract

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 NaV1.7 subtype of voltage-gated sodium (NaV) 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 Phlogiellus genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) NaV1.7 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 NaV1.7-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 hNaV subtypes, as well as two cardiac targets, the hCaV1.2 and hKV11.1 subtypes of voltage-gated calcium (CaV) and potassium (KV) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNaV1.1–1.3 and 1.5–1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNaV1.4 and 1.8, hCaV1.2 and hKV11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of NaV1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the NaV1.7 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 NaV1.7 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.

Fifteen years of NaV1.7 channels as an analgesic target: Why has excellent in vitro pharmacology not translated into in vivo analgesic efficacy?

“…No neurotoxicity found (Deuis et al, 2017;Goncalves et al, 2019;Nicolas et al, 2019) JzTx 34 610 Effective in reducing formalin-induced, acetic acid-induced, and thermal pain in mice. As efficacious as morphine (i.p.…”

Anti-inflammatory activities of arthropod peptides: a systematic review

“…Phlotoxin 1 (Ph1Tx1) is a 34-residue toxin purified from Phlogiellus spider venom, a promising antinociceptive peptide with a high affinity for Pav [ 128 ]. The crude venom of Phoneutria nigriventer (armed spiders), besides its antineoplastic activity, can suppress the IFN-γ release and increase the expression of the anti-inflammatory cytokine IL-10.…”

Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

“…Database search of the monoisotopic oxidized peptide masses of the whole and fractionated spider venom from P. bundokalbo showed that all the peptide masses were not similar to masses of 3 isoforms of peptides from Phlogiellus sp. recorded on Arachnoserver which are µ-theraphotoxin-Phlo1a (4103.79 Da), µ-theraphotoxin-Pho1b (4137.78 Da), and µ-theraphotoxin-Phlo2a (N-terminal fragment) (3276.33 Da) [ 45 , 55 , 56 ]. On the other hand, some of the peptides present in the whole and fractionated venom have similar masses present in the database from other species of spider that are related to P. bundokalbo by family and order as shown in Table 2 .…”