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Spider polypeptide toxin with Nav1.9 specific activation effect and application of spider polypeptide toxin

An activation, spider technology, applied in the field of peptides, can solve problems such as self-harm, bone fractures, no therapeutic drugs and treatment strategies

Active Publication Date: 2020-07-31
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Clinically, loss-of-function mutations in Nav1.7, a voltage-gated sodium channel subtype distributed in peripheral nociceptors, lead to congenital inability to pain (CIP), which cannot distinguish between sharp and frustrating stimuli , often resulting in self-harm or broken bones and accidental death
It is a very serious pain disorder for which there are still no effective treatment drugs and treatment strategies

Method used

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  • Spider polypeptide toxin with Nav1.9 specific activation effect and application of spider polypeptide toxin
  • Spider polypeptide toxin with Nav1.9 specific activation effect and application of spider polypeptide toxin
  • Spider polypeptide toxin with Nav1.9 specific activation effect and application of spider polypeptide toxin

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Experimental program
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Effect test

Embodiment 1

[0066] HpTx1 restores pain response in Nav1.7 knockout mice

[0067] We separated the white-fronted tall spider (H. venatoria) venom by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Chromatography (figure 1 Middle a panel) was purified again to obtain a polypeptide toxin HpTx1 (system name κ-sparatoxin-Hv1a), which was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and its molecular weight was 3910.8Da. Sequence comparison with polypeptide toxins of known structure found that HpTx1 showed certain sequence similarities, especially the conserved cysteine ​​pattern, which is the pattern adopted by most spider polypeptide toxins-inhibitor cystine knot ( ICK) motif ( figure 1 Middle b).

[0068] Injection of 10 μM HpTx1 into the hind paws of Nav1.7 knockout (Nav1.7-KO) mice or control littermate wild-type mice (fNav1.7) triggered strong nociceptive responses, such as licking or biting ...

Embodiment 2

[0070] HpTx1 enhances the excitability of DRG neurons in Nav1.7-KO mice

[0071] Current-clamp recordings were used to investigate whether HpTx1 directly activates sensory neurons. To detect the effect of HpTx1 on the action potential (AP) burst of small-diameter ( figure 2 As shown in panel a, 0.75 μM HpTx1 shifted the RMP by about 2.4 mV in the direction of depolarization (Table 1). At the same time, 0.75μM HpTx1 also significantly reduced the action potential current threshold, about 9.3pA ( figure 2 Middle b panel, Table 1). Of the 30 DRG neurons tested, current thresholds decreased in 15 neurons (50.0%), remained unchanged in 40%, and increased in 10% in the presence of HpTx1. However, no significant change in action potential amplitude was observed in the presence of HpTx1 ( figure 2 c, Table 1); Among the 15 neurons sensitive to HpTx1, the frequency of action potential bursts in 8 neurons was significantly increased ( figure 2 Middle d panel). Notably, the inpu...

Embodiment 3

[0077] HpTx1 inhibits Nav1.7 and activates Nav1.9

[0078] HpTx1-induced pain responses were not different between wild-type (WT) and Nav1.7-KO mice, suggesting that HpTx1-induced pain responses should not be related to Nav1.7. However, HpTx1 inhibited hNav1.7 currents expressed in HEK 293T cells, IC 50 The value is 0.51±0.12μM ( image 3 Middle panel a). However, 1 μM HpTx1 did not change the steady-state activation and inactivation curves of Nav1.7 channels ( image 3 Middle b panel, Table 2). In addition, the effect of HpTx1 on (tetrodotoxin-sensitive) TTX-S Nav channels in small-diameter DRG neurons of WT mice was detected, and it was found that 1 μM HpTx1 inhibited 62.9±7.8% of TTX-S Nav currents ( image 3 middle c). Then besides Nav1.7, TTX-S type Nav1.6 is also expressed in small diameter DRG neurons. Indeed, HpTx1 has inhibitory activity on Nav1.6 current, IC 50 The value is 5.63 ± 0.13 μM, but it is nearly 10 times weaker than that of Nav1.7. Furthermore, Vas...

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Abstract

The invention relates to spider polypeptide toxin with a Nav1.9 specific activation effect. The spider polypeptide toxin controls a sodium channel subtype Nav1.9 through a specific activation of a voltage gate, so as to recover the pain feeling of a patient with pain insensitivity. Experiments prove that HpTx1 is unique to other channels for enhancing the Nav1.9 function, and animal experiments show that the spider polypeptide toxin can enhance related congenital painless pain behaviors caused by Nav1.7 loss and recover mechanical pain sensitivity and the like, so that clinical application totreat pain insensitivity diseases, especially congenital painless diseases related to Nav1.7. Therefore, the invention also provides an application of the polypeptide toxin in preparation of a medicine for treating congenital pain deficiency, especially an application in preparation of a medicine for treating Nav1.7 related congenital painless diseases. In addition, the HpTx1 is a good Nav1.9 channel tool reagent.

Description

technical field [0001] The invention relates to a polypeptide, in particular to a spider polypeptide toxin with Nav1.9 specific activation function and application thereof. Background technique [0002] For each individual, pain is a very unpleasant feeling, and pain is also one of the common clinical symptoms. Currently, nearly 20% of the world's population suffers from chronic pain all year round. In peripheral sensory neurons, the other two subtypes Nav1.8 and Nav1.9 specifically co-expressed with Nav1.7 subtype Nav1.7 have also been proved to be key targets for regulating pain. Because of this, these ion channels also It has become an important target for the development of drugs for pain-related diseases. The three channels Nav1.7, Nav1.8 and Nav1.9 are the basis for the generation and propagation of neuronal action potentials, and they are jointly involved in the pain signaling pathway. However, pain is part of the body's defense system, always reminding us to avoid...

Claims

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Application Information

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IPC IPC(8): C07K14/435A61K38/17A61P29/00
CPCC07K14/43518A61P29/00A61K38/00
Inventor 刘中华周熙陈敏芝
Owner HUNAN NORMAL UNIVERSITY
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