Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Photoluminescent gold nanoclusters (Au NCs) were synthesized in one step in aqueous conditions using a mixture of glutathione and (mono or multivalent) glutathione modified arginine peptides. By controlling the ratio of co-ligands, we investigated how the multivalency and the amount of arginines influenced the growth of Au NCs, their surface chemistry, their colloidal stability, and their optical properties. We demonstrated using two-dimensional nuclear magnetic resonance spectroscopy that the organisation of the ligand on the Au surface was composed by an inner rigid layer and an outer flexible part via inter-and/or intra-ligand spatial proximity. This directly impacted the structure of the Au NCs, as confirmed by gel electrophoresis, high transmission electron microscopy, diffusion ordered spectroscopy and mass spectrometry. Increasing arginine content also induced an increase of positive surface charge and an enhancement of the near infrared emission intensity at ~670 nm with quantum yield up to 10% validating the significant influence of the ligands to the optical properties of Au NCs.

Background and Purpose Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the Nav1.7 voltage‐dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its Nav channel targets. Experimental Approach We took advantage of the high affinity properties of the peptide, combined to its slow off rate, to design a number of new tagged analogues useful for imaging and biochemistry purposes. We used high‐throughput automated patch‐clamp to identify the analogues best matching the native properties of ProTx II and validated them on various Nav‐expressing cells in pull‐down and cell distribution studies. Key Results Two of the produced ProTx II analogues, Biot‐ProTx II and ATTO488‐ProTx II, best emulate the pharmacological properties of unlabelled ProTx II, whereas other analogues remain high affinity blockers of Nav1.7. The biotinylated version of ProTx II efficiently works for the pull‐down of several Nav isoforms tested in a concentration‐dependent manner, whereas the fluorescent ATTO488‐ProTx II specifically labels the Nav1.7 channel over other Nav isoforms tested in various experimental conditions. Conclusions and Implications The properties of these ProTx II analogues as tools for Nav channel purification and cell distribution studies pave the way for a better understanding of ProTx II channel receptors in pain and their pathophysiological implications in sensory neuronal processing. The new fluorescent ProTx II should also be useful in the design of new drug screening strategies.

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