Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNa_v1.7 could be inhibited by rHWTX-I and the IC₅₀ value was 419 nmol/L.

2022-08-01·Current Neuropharmacology2区 · 医学

Animal Venom Peptides Cause Antinociceptive Effects by Voltage-gated Calcium Channels Activity Blockage

2区 · 医学

Article

作者: Trevisan, Gabriela ; Oliveira, Sara Marchesan

:Pain is a complex phenomenon that is usually unpleasant and aversive. It can range widely in intensity, quality, and duration and has diverse pathophysiologic mechanisms and meanings. Voltage-gated sodium and calcium channels are essential to transmitting painful stimuli from the periphery until the dorsal horn of the spinal cord. Thus, blocking voltage-gated calcium channels (VGCCs) can effectively control pain refractory to treatments currently used in the clinic, such as cancer and neuropathic pain. VGCCs blockers isolated of cobra Naja naja kaouthia (α-cobratoxin), spider Agelenopsis aperta (ω-Agatoxin IVA), spider Phoneutria nigriventer (PhTx3.3, PhTx3.4, PhTx3.5, PhTx3.6), spider Hysterocrates gigas (SNX-482), cone snails Conus geographus (GVIA), Conus magus (MVIIA or ziconotide), Conus catus (CVID, CVIE and CVIF), Conus striatus (SO- 3), Conus fulmen (FVIA), Conus moncuri (MoVIA and MoVIB), Conus regularis (RsXXIVA), Conus eburneus (Eu1.6), Conus victoriae (Vc1.1.), Conus regius (RgIA), and spider Ornithoctonus huwena (huwentoxin-I and huwentoxin-XVI) venoms caused antinociceptive effects in different acute and chronic pain models. Currently, ziconotide is the only clinical used N-type VGCCs blocker peptide for chronic intractable pain. However, ziconotide causes different adverse effects, and the intrathecal route of administration also impairs its use in a more significant number of patients. In this sense, peptides isolated from animal venoms or their synthetic forms that act by modulating or blocking VGCCs channels seem to be a relevant prototype for developing new analgesics efficacious and well tolerated by patients.

2022-05-01·The Journal of Physiology2区 · 医学

Non‐canonical endogenous expression of voltage‐gated sodium channel Na_V1.7 subtype by the TE671 rhabdomyosarcoma cell line

2区 · 医学

Article

作者: Mohammed Latif, Liaque ; Aziz, Muhammad Y. A. ; Duce, Ian R. ; Mellor, Ian R. ; Wall, Richard J. ; Ngum, Neville M.

AbstractThe human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage‐sensitive sodium currents in these cells, the specific voltage‐gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT‐PCR), western blot and whole‐cell patch clamp electrophysiology were performed. qRT‐PCR profiling revealed that expression of the SCN9A gene was ∼215‐fold greater than the SCN4A gene and over 400‐fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of −31.9 ± 1.1 and −69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50 = 22.3 nm). They were also very susceptible to the NaV1.7 specific blockers Huwentoxin‐IV and Protoxin‐II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non‐canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost‐effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain.Key points

Undifferentiated TE671 cells produce a voltage‐sensitive sodium current when depolarised.

The voltage‐gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown.

Through qRT‐PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain.

The TE671 cell line is, therefore, a very easy to maintain and cost‐effective model to study NaV1.7‐targeting drugs.

100 项与虎纹镇痛肽相关的药物交易

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