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Phlotoxin
Phlotoxin (phltx1) is a neurotoxin from the venom of a genus of tarantula (Phlogiellus) that targets mostly voltage-sensitive sodium channels and mainly Nav1.7. The only non-sodium voltage-sensitive channel that is slightly inhibited by Phlotoxin is the Kv3.4. Nav1.4 and Nav1.6 seem to be Phlotoxin-1 sensitive to some extent as well.

Etymology:
Another name for phlotoxin is µ-TRTX-Pspp-1 : µ for NaV channel inhibition, then TRTX refers to theraphotoxin which refers to a group of toxins found in the Theraphosidae family.

Source:
Phlogiellus spider, is a genus of tarantulas. Its venom, which includes several neurotoxic peptides like phlotoxin, targets diverse ion channels and chemical receptors.Phlotoxin was first purified, characterized and sequenced from Phlogiellus sp.,  Theraphosidae  Selenocosmiinae (endemic to Papua New Guinea) sequenced.

Chemistry:
Phlotoxin-1 (PhlTx1), weighing around 4058.83 Da, is identified by a 34-amino acid sequence featuring three disulfide bridges and organized based on the ICK (Inhibitory Cysteine Knot) architectural motif which is effective for its structural stabilization as three disulfide bonds are structured in a manner where two of them combine to create a circular arrangement, through which the third disulfide bond passes. It is classified to be a member of the NaV channel spider toxin (NaSpTx) family 1. The structure of phlotoxin comprises six cysteine residues forming an inhibitory cysteine-knot (ICK) architecture fold, with amidation occurring at the C-terminus. “Cys2-Cys17, Cys9-Cys22, Cys16-Cys29” disulfide bridge organization. The proximity of Cys 16 and 17 makes it challenging to be synthesized even though the Phlotoxin is commercially available.

Homology:
PhlTx1 does not closely resemble known toxin peptides. The sequence similarity varies from 24% to 59% at most, suggesting limited homology with previously identified peptides. The closest match regarding inhibition IC50 for PhlTx1 is found in the NaSpTx family to HnTx-III or HwTx-I. It is basically classified under the NaSpTx family, due to the presence of disulfide bridges. PhlTx1 is categorized within the NaSpTx-1 family primarily because of its disulfide bridges. Notably, the inclusion of three proline residues (Pro11, Pro18, and Pro27) introduces the potential for trans/cis isomerization. This dynamic property can influence the precise formation of secondary structures and the correct alignment of disulfide bridges, thereby impacting the overall structural integrity of the toxin.

Target:
In examining the effects of PhlTx1 on the sodium channel Nav1.7/β1, it appears to share similarities with TTX (tetrodotoxin). Both PhlTx1 and TTX exhibit a capacity to block the channel pore, resulting in a noticeable decrease in sodium currents. Moreover, the behavior of the channel, as reflected in gating parameters, remains largely unaffected by the presence of PhlTx1. This observation suggests a comparable behavior between PhlTx1 and TTX in modulating the function of Nav1.7/β1 channels. The IC50 for PhlTx1 to inhibit Nav1.7 is 39 +/- 2 nM.

The PhlTx1 affects all hNav channels to a different degree except hNav1.8. There is also poor selectivity of PhlTx1 towards the hNaV1.1 and 1.3 subtypes, this poor selectivity may actually be an advantage since these subtypes are also involved in pain pathways. However, the poor selectivity towards the hNaV1.5 and 1.6 subtypes may be associated with in vivo cardiac and neuromuscular side effects, respectively, which could limit its potential use as an analgesic molecule. It also has shown a high affinity towards hNav1.7, which made it interesting to do in vivo studies that revealed that PhlTx1 exhibits potent analgesic effects in a mouse model of NaV1.7-mediated pain. The study also reported that no sign of cardiac or neuromuscular side effects were detected during experiments with PhlTx1 in animal models.

The amino acids which are critical for binding of the hNaV1.7 subtype are identified by their substitution with alanine. When Tryptophan at position 24, Lysine at position 25 and Tyrosine at position 26 are replaced with alanine, there is a complete loss of affinity. This highlights the critical role of these amino acids in the binding process to Nav1.7. Other substitutions, like Alanine at position 1, Serine at position 8, Lysine at position 12 or 15, result in a slight change (less than 2.8-fold) in variant affinity. While substituting Aspartate at position 7 leads to an increase in variant affinity (IC50 = 47.0 ± 40.9).

Therapeutic use:
Phlotoxin-1 (PhlTx1) has demonstrated selectivity in inhibiting the voltage-gated sodium channel NaV1.7. Its potential as an antinociceptive agent became apparent when a loss-of-function mutation in the NaV1.7 gene resulted in a congenital inability to perceive pain. Notably, these peptides do not independently exhibit antinociceptive effects; however, when co-administered with exogenous opioids, they bring about analgesia, allowing for a significant reduction in opioid dosage. The mechanism underlying the synergistic effect of opioid receptor agonists with selective NaV1.7 inhibitors remains unknown, but this discovery presents a novel approach to pain management. The primary method for evaluating this property involves the formalin test.

External Links:
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/inhibitor-cystine-knot

https://alphafold.ebi.ac.uk/entry/P0DM14

https://www.uniprot.org/uniprot/P0DM14