The immune system and the impact of zinc during aging

The trace element zinc is essential for the immune system, and zinc deficiency affects multiple aspects of innate and adaptive immunity. There are remarkable parallels in the immunological changes during aging and zinc deficiency, including a reduction in the activity of the thymus and thymic hormones, a shift of the T helper cell balance toward T helper type 2 cells, decreased response to vaccination, and impaired functions of innate immune cells. Many studies confirm a decline of zinc levels with age. Most of these studies do not classify the majority of elderly as zinc deficient, but even marginal zinc deprivation can affect immune function. Consequently, oral zinc supplementation demonstrates the potential to improve immunity and efficiently downregulates chronic inflammatory responses in the elderly. These data indicate that a wide prevalence of marginal zinc deficiency in elderly people may contribute to immunosenescence

In vivo evaluation of antinociceptive effects of cyriotoxin-1a, the first toxin purified fromCyriopagopus schioedtei spider venom

International audienceOver the past two decades, animal venom toxins have been widely explored as an original source of new antinociceptive drugs directed towards the Nav1.7 subtype of voltage-gated sodium channels. This subtype, expressed in afferent sensitive fibers and more particularly in dorsal root ganglia (DRG) neurons, has been validated as an antinociceptive target of choice by human genetic evidence such as congenital insensivity to pain [1]. High throughput screening of Smartox biotechnology company collection venoms, using automated patch-clamp (QPatch HTX, Sophion Biosciences) on HEK cells overexpressing human Nav subtypes, pointed out a new inhibitory cysteine knot (ICK) toxin, named cyriotoxin-1a (CyrTx-1a). In DRG neurons isolated from adult mice, the peptide preferentially inhibited with high affinity tetrodotoxin (TTX)-sensitive Na current (IC50 = 170 nM), flowing through mainly the Nav1.7 subtype, compared to TTX-resistant Na current (IC50 =108 μM), flowing through Nav1.8 and Nav1.9 subtypes. In addition, the peptide exhibited nanomolar range affinity for Nav1.7, Nav1.1-1.3 and NaV1.6 subtypes and micromolar range affinity for Nav1.5 and Nav1.4 subtypes. The hot plate and von Frey pain assays showed that mice injected with 367 μg/kg of CyrTx-1a were more resistant to pain than animals injected with the vehicle (PBS). However, compound muscle action potential (CMAP) recordings after toxin injection revealed a relative narrow therapeutic window w/o side-effects. In conclusion, the pharmacological profile of CyrTx-1a is of great interest since it leads to further engineering studies aimed to improve its selectivity for optimizing the use of this peptide as an antinociceptive therapeutics.[1].Vetter et al. (2016) Nav1.7 as a pain target – from gene to pharmacology. Pharmacology and Therapeutics 172, 73-100

D peptide, a promising toxin as potential antinociceptive agent targeting the Nav1.7 subtype of voltage-gated sodium channels

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Multiscale functional approaches to evidence high affinity blockade of Nav1.6 channel subtype by huwentoxin-IV spider peptide

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Direct evidence for high affinity blockade of Nav1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches

International audienceThe Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of NaV1.7 subtype of voltage-gated sodium (NaV) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and NaV1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of NaV1.6, compared to NaV1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNaV1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the NaV1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity

A multiscale functional approach to assess pharmacologocal interactions between huwentoxin-IV spider peptide and sodium channel subtypes

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Translational engagement of LPA1_{1} receptor in skin fibrosis: from dermal fibroblasts of patients with scleroderma to Tsk1 mouse

BACKGROUND AND PURPOSE: Genetic deletion and pharmacological studies suggest a role for lysophosphatidic acid receptor-1 (LPA$_{1}$ receptor) in fibrosis. We investigated the therapeutic potential in systemic sclerosis (SSc) of a new orally active selective LPA$_{1}$ receptor antagonist using dermal fibroblasts from patients and animal model of skin fibrosis. EXPERIMENTAL APPROACH: Dermal fibroblast and skin biopsies from SSs patients were used. Myofibroblast differentiation, gene expression and cytokine secretion were measured following LPA and/or SAR100842 treatment. Therapeutic effect of SAR100842 was assessed in the tight skin mouse model-1 (Tsk1). KEY RESULTS: SAR100842 is equipotent against various LPA isoforms. SSc dermal fibroblasts and skin biopsies expressed high levels of LPA$_{1}$ receptor. The LPA functional response (Ca$^{2+}$ ) in SSc dermal fibroblasts was fully antagonized with SAR100842. LPA induced myofibroblast differentiation in SSc dermal and IPF lung fibroblasts and the secretion of inflammatory markers and activated Wnt markers. Results from SSc dermal fibroblasts mirror those obtained in a mouse Tsk1 model of skin fibrosis. Using a therapeutic protocol, SAR100842 consistently reversed dermal thickening, inhibited myofibroblast differentiation and reduced skin collagen content. Inflammatory and Wnt pathway markers were also inhibited by SAR100842 in the skin of Tsk1 mice. CONCLUSION: The effects of SAR100842 on LPA -induced inflammation and on mechanisms linked to fibrosis like myofibroblast differentiation and Wnt pathway activation indicate that LPA$_{1}$ receptor activation plays a key role in skin fibrosis. Our results support the therapeutic potential of LPA$_{1}$ receptor antagonists in systemic sclerosis

Venoms in drug discovery: a case study on voltage-gated channels

International audienceAmong all chemical modalities available in ion channel drug discovery, venoms are very attractive for identifying potent and selective compounds for almost all subunits of this therapeutic target family. Here, we describe a highthroughput approach for the identification and characterization of voltage-gated modulators from a large venom collection. Using this strategy, we were able to describe different mechanism of actions of subfractions and in particular antagonists of the Nav1.7 channel subunit. These results are illustrated with the characterization of the first peptide isolated from the Cyriopagopus schioedtei spider. This peptide, named cyriotoxin-1a (CyrTx-1a), is composed of 33 amino acids, contains the ICK structural motif and inhibited hNav1.1-1.3 and 1.6-1.7 in the low nanomolar range, compared to the micromolar range for hNaV1.4-1.5 and 1.8. In agreement, CyrTx-1a was 635 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons. Within a narrow therapeutic window of concentration, CyrTx-1a exhibited an analgesic-like effect in mice by increasing reaction time in the hot plate assay and decreasing tactile sensitivity in automated plantar von Frey experiments. Finally, in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. This pharmacological profile paves the way for further engineering studies aimed at optimizing the potential antinociceptive properties of CyrTx-1a. We will conclude by discussing these results in the context of an integrated drug discovery research platform

Fluorescent‐ and tagged‐protoxin II peptides: potent markers of the Na v 1.7 channel pain target

International audienceBackground 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