Identification of a novel peptidic specific blocker of Nav1.7 sodium channel subtype with analgesic properties

Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique.The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests

Identification d'un nouveau bloqueur peptidique spécifique du canal sodique Nav1.7 avec des propriétés analgésiques

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests.Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique

Identification d'un nouveau bloqueur peptidique spécifique du canal sodique Nav1.7 avec des propriétés analgésiques

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, and therefore is an interesting drug target to develop innovative analgesic compounds for improved chronic pain management. Despite recent advances in the identification of small organic molecules targeting the Cav3.x/T-type calcium channel family, to date specific Cav3.2 inhibitors remains to be identified. Spider venoms proved to contain a large diversity of neurotoxins including gating modifiers of calcium channels. Using recombinant Cav3.2 channels, we performed a screening of a Tarantula venom library and identified a new 28 amino acid peptide (Psp3Tx1). The synthetic form of the peptide was used to determine its selectivity profile over a panel of closely related members of the voltage gated calcium (Cav) and sodium (Nav) channels. The peptide proved to be selective for the Nav1.7 channel (largely validated target in the context of pain pathologies) with an additional effect on Cav3.2 at more elevated doses. In vivo in mice, the peptide demonstrated to be an efficient analgesic molecule with anti hyperalgesic and antiallodynic properties in the context of neuropathic pain. This peptide also possess analgesic properties in a context of spontaneous pain induced by a Nav1.7 agonist. The results presented in this thesis are encouraging for the setup of a project taking Psp3Tx1 into clinical tests.Les canaux calciques de type T Cav3.2 sont des régulateurs clés des fonctions sensorielles, et de fait sont également des cibles intéressantes pour le développement de nouveaux composés analgésiques pour le traitement et le management de la douleur. Malgré de récentes avancées dans l’identification de petites molécules organiques ciblant la famille des canaux Cav3.x/Type T, il reste encore à identifier un inhibiteur spécifique de Cav3.2. Le venin d’araignées contient une large diversité de neurotoxines incluant des modificateurs de gating des canaux calciques. En utilisant des canaux calciques recombinants, nous avons procédé à un criblage d’une bibliothèque de venins et avons identifié un nouveau peptide de 28 acides aminés (Psp3Tx1). La forme synthétique de ce peptide a été utilisé pour déterminer son profil de selectivité sur un panel de membres proches de la famille des canaux calciques (Cav) et sodiques (Nav) dépendants du voltage. Le peptide est sélectif pour le canal Nav1.7 (une cible largement validée dans le contexte des pathologies de la douleur) avec un effet complémentaire sur Cav3.2 à de fortes doses. In vivo chez la souris, le peptide possède des propriétés analgésiques avec des effets anti-hyperalgésiques et anti-allodyniques dans un contexte de douleur neuropathique. Ce peptide possède également un pouvoir analgésique dans un contexte de douleur spontanée induit par un agoniste des canaux Nav1.7. Les résultats présentés dans cette thèse sont encourageants pour la mise en place d’un projet amenant Psp3Tx1 au niveau clinique

Balanus Da 1778

Genus <i>Balanus</i> Da Costa, 1778 <p> <b>Type species.</b> <i>Lepas balanus</i> Linnaeus, 1758, by original designation.</p> <p> <b>Diagnosis.</b> Wall of six plates, tubiferous, with one row of major tubes with transverse septa, at least when young; radii solid with transverse teeth; alae not cleft; lateral margin of sheath not extending over adjacent ala, basis solid or tubiferous in single layer. Scutum with adductor ridge, when present, weak. Tergum with spur with gentle change in direction of growth lines, spur and furrow margins coincident; basicarinal angle with well-developed depressor muscle crests limited to border (after Carriol 2008).</p>Published as part of <i>Carriol, René-Pierre, Cahuzac, Bruno & Lesport, Jean-François, 2011, New species of Acasta and Balanus (Balanoidea: Acastinae, Balaninae) from the Early Miocene (Burdigalian) of the Aquitaine Basin, France, pp. 60-68 in Zootaxa 3109</i> on page 66, DOI: <a href="http://zenodo.org/record/201918">10.5281/zenodo.201918</a&gt

Acasta martillacensis Carriol, sp. nov.

<i>Acasta martillacensis</i> Carriol sp. nov. <p>Figs. 2A–D, 3</p> <p> <b>Type locality.</b> Martillac ditch section, near 'Couvent de la Solitude', Département Gironde, Aquitaine Basin, southwest France.</p> <p> <b>Type stratum.</b> Early Burdigalian, fine grey-blue sands in the lower portion of the section exposed, level GA 39191, “Sables à Mactres”.</p> <p> <b>Material examined.</b> Holotype (no. 85-12-68, Cahuzac Collection), a shell (basis with wall plates), retaining the right scutum. Paratypes, (no. 85-12-69, Lesport Collection) a rostrum; (no. 85-12-70, Lesport Collection) half a basis with carina, a carinolateral, lateral and half rostrum.</p> <p> <b>Additional material</b> (Lesport Collection). 2 rostra, 2 carinae, 1 basis, 1 fragmentary basis with a partial lateral and carinolateral.</p> <p> <b>Measurements.</b> Holotype: opercular aperture, carino-rostral diameter 3.5 mm; carina at mid-height 4.4 mm; rostrum at mid-height 3.9 mm, sheath height 1.9 mm; basis, carino-rostral diameter 4.1 mm; scutum, occludent margin 3.1 mm, articular margin 2.0 mm, articular ridge 1.4 mm. Paratype no. 85-12-69: rostrum at mid-height 6.3 mm. Paratype no. 85-12-70: basis, carino-rostral diameter 7.0 mm, opercular aperture, carino-rostral diameter 5.5 mm. Additional material: 2 rostra, at mid-height 5.1 and 6.6 mm, respectively; 2 carinae, at mid-height 5.6 and 7.1 mm, respectively; basis, larger diameter 6.0 mm.</p> <p> <b>Diagnosis.</b> Shell with internal longitudinal ribs; cellular space between sheath and wall; carinal sheath adhering to the wall in the centre of its basal edge; basis cup shaped; scutum externally with radial striae, and internally with adductor ridge, not prominent.</p> <p> <b>Description.</b> Shell white, occasionally with pink hue towards apex and on radii (original coloration visible on holotype). Parietes internally with well-developed ribbing extending up to sheath or nearly so. Between ribs, in parts of plate where they are close, fine longitudinal spaces present. These tubes do not show an aperture between the ribs near basal margin of plates (see Fig. 3); only in broken plates can tubes be demonstrated. Parietes externally with growth ridges crossed by very fine longitudinal ribs, and with, hollow, upward-curving spines sloping downwards, spines occasionally bifurcated. Radii and alae not very wide, upper edge oblique, sutural edge of radii with striations. Sheath broad, occupying about half length of compartment, transversally striated with basal edge free from wall except in carina, where central part of basal edge turns down, adhering to wall. Cellular space, secondarily calcified, between sheath and wall. Basis cup-shaped, calcareous, non porous, concentrically ringed outside but smooth inside, with margin bearing small pits for reception of teeth formed by ends of internal parietal ribs. Scutum wide, taller than wide, slightly bowed between apex and basal margin; occludent margin slightly concave, tergal margin straight, basal margin convex; exterior ornamented by growth increments crossed by incised radial striae; interior, with articular ridge not prominent barely extending over the tergal margin, and with adductor ridge not prominent, of same length as tergal edge of adductor muscle pit; lateral depressor muscle scar rounded, rather deep; adductor muscle pit shallow, rather wide with its posterior extremity not clearly discernible.</p> <p> <b>Etymology.</b> In reference to Martillac, Aquitaine Basin (southwest France).</p> <p> <b>Remarks.</b> The specimens of <i>Acasta martillacensis</i> <b>sp. nov.</b> have ribbed parietal plates as do other numerous species of <i>Acasta</i>. No apertures of the tubes are visible between these ribs near the basal margin of the plate. It is along breaks of certain specimens that these tubes appear and these specimens, which seem rather old in view of their size, are only partially tubiferous. Tubes are present only in particular parts of the plates, in the parts where ribs are numerous and in contact, as if the ribs by contacting had closed a longitudinal space between them. These tubes are not the same that those present in <i>Perforatus perforatus</i> and are different to those in <i>Balanus crenatus</i>. They are not tubes in the sense we usually understand and the presence of solid paries remains a characteristic of <i>Acasta</i>.</p> <p> <b>Affinities.</b> Among the 26 extant species retained in the genus <i>Acasta</i> by Kolbasov (1993), one species has fossil representatives: <i>Acasta spongites</i>, the extant species from the Atlantic and the Mediterranean Sea, which has been described from the Pliocene of France (De Alessandri 1907) and Italy (Moroni 1952). There are eight extinct species (Carriol 2008). The <i>Acasta</i> with fossil representatives, except <i>A. vesiculosa</i> Carriol, 2008 (see complete description in Schneider <i>et al.</i> 2009), may be distinguished from <i>Acasta martillacensis</i> <b>sp. nov.</b> in the lack of a vesicular sheath. <i>Acasta martillacensis</i> <b>sp. nov.</b> is close to <i>A. vesiculosa</i> in having the vesicular sheath and a carinal sheath adhering to the wall in the centre of its basal edge. However, the former can be distinguished by the internal ribbing of the wall, its cup-shaped basis and its scutum, in which the adductor ridge is not prominent. The claim that <i>A. vesiculosa</i> from the middle Miocene of France (Carriol 2008) showed internal ribbing of the wall is erroneous, due to the poor preservation of a small set of specimens available.</p>Published as part of <i>Carriol, René-Pierre, Cahuzac, Bruno & Lesport, Jean-François, 2011, New species of Acasta and Balanus (Balanoidea: Acastinae, Balaninae) from the Early Miocene (Burdigalian) of the Aquitaine Basin, France, pp. 60-68 in Zootaxa 3109</i> on pages 63-65, DOI: <a href="http://zenodo.org/record/201918">10.5281/zenodo.201918</a&gt

Balanus aculeatus Carriol, sp. nov.

<i>Balanus aculeatus</i> Carriol sp. nov. <p>Fig. 2 E–H</p> <p> <b>Type locality.</b> Saucats (Le Péloua), Département Gironde, Aquitaine Basin, southwest France.</p> <p> <b>Type stratum.</b> Early Burdigalian, shell-rich, rusty, coarse carbonate sands, with rounded clasts of reefal corals.</p> <p> <b>Material examined.</b> Holotype: no. 19-12-23 (Lesport Collection), a carina with a fragment of basis in connection attached.</p> <p> <b>Measurements.</b> Holotype: mid-height of parietes 29.3 mm.</p> <p> <b>Diagnosis.</b> Paries exteriorly with strong, tubular spines.</p> <p> <b>Description.</b> Plate straight, relatively tall, folded under an angle of 60 degrees at sheath level. Paries outer surface brown, with fine growth ridges crossed by very fine longitudinal ridges in superior 3/4, and longitudinal rows of strong, tubular spines. Paries tubiferous with 1 row of tubes with transverse septa; primary longitudinal septa, 27 in number, with lateral denticules. Sheath with growth ridges, lacking longitudinal ribs, occupying more than upper 1/2 of interior of plate; lower edge of sheath free of shell wall, with deep cavity beneath. Alae not cleft, with summits almost horizontal. Radii with transverse teeth on sutural edge smooth (characteristic deduced from traces left by sutural edge of radii of carinolateral on paries). Basis flat, calcified, tubiferous, single layered; tubes small with transverse septa.</p> <p> <b>Etymology.</b> Alluding to the calcareous spines of the paries.</p> <p> <b>Affinities.</b> <i>Balanus nodulus</i> Carriol, 2008 is the only other balanine with calcareous projections. The new species differs from <i>B. nodulus</i> in having both the tubes of the parietes and of the basis with transverse septa and by the parietal projections, which are strong, tubular spines, rather than small protuberances.</p>Published as part of <i>Carriol, René-Pierre, Cahuzac, Bruno & Lesport, Jean-François, 2011, New species of Acasta and Balanus (Balanoidea: Acastinae, Balaninae) from the Early Miocene (Burdigalian) of the Aquitaine Basin, France, pp. 60-68 in Zootaxa 3109</i> on page 66, DOI: <a href="http://zenodo.org/record/201918">10.5281/zenodo.201918</a&gt

Acasta Leach 1817

Genus <i>Acasta</i> Leach, 1817 <p> <b>Type species.</b> <i>Lepas spongites</i> Poli, 1791, by original designation.</p> <p> <b>Diagnosis.</b> Basis deeply cup shaped, rarely flat, with circular margin, often bearing small teeth; internal surface smooth, without six radial ribs. Wall plates usually with longitudinal, internal ribs; in some species, wall plates separated by splits or windows covered by delicate membrane. Carinolaterals developed normally. Scutum with or without radial striae. Cirral armature variable, segments of anterior ramus of cirrus IV bearing hooks, or thorns, or lacking either (after Kolbasov 1993).</p>Published as part of <i>Carriol, René-Pierre, Cahuzac, Bruno & Lesport, Jean-François, 2011, New species of Acasta and Balanus (Balanoidea: Acastinae, Balaninae) from the Early Miocene (Burdigalian) of the Aquitaine Basin, France, pp. 60-68 in Zootaxa 3109</i> on page 63, DOI: <a href="http://zenodo.org/record/201918">10.5281/zenodo.201918</a&gt

Evaluation of the Spider (Phlogiellus genus) Phlotoxin 1 and Synthetic Variants as Antinociceptive Drug Candidates

International audienceOver the two last decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. At present, approximately 20 potential analgesic toxins, mainly from spider venoms, are known to inhibit with high affinity the Na V 1.7 subtype of voltage-gated sodium (Na V) channels, the most promising genetically validated antinociceptive target identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a Phlogiellus genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) Na V 1.7 subtype. The synthetic homologue of PhlTx1 was generated and equilibrated between two conformers on reverse-phase liquid chromatography and exhibited potent analgesic effects in a mouse model of Na V 1.7-mediated pain. The effects of PhlTx1 and 8 successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNa V subtypes, as well as two cardiac targets, the hCa V 1.2 and hK V 11.1 subtypes of voltage-gated calcium (Ca V) and potassium (K V) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNa V 1.1-1.3 and 1.5-1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNa V 1.4 and 1.8, hCa V 1.2 and hK V 11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of Na V 1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the Na V 1.7 subtype was at least five times higher than that of the wild-type peptide. Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure-activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the Na V 1.7 subtype, but without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates

Reduced recruitment of 53BP1 during interstrand crosslink repair is associated with genetically inherited attenuation of mitomycin C sensitivity in a family with Fanconi anemia

The Fanconi anemia (FA) pathway is implicated in the repair of DNA interstrand crosslinks (ICL). In this process, it has been shown that FA factors regulate the choice for DNA double strand break repair towards homologous recombination (HR). As this mechanism is impaired in FA deficient cells exposed to crosslinking agents, an inappropriate usage of non-homologous end joining (NHEJ) leads to the accumulation of toxic chromosomal abnormalities. We studied a family with two FANCG patients and found a genetically inherited attenuation of mitomycin C sensitivity resulting in-vitro in an attenuated phenotype for one patient or in increased resistance for two healthy relatives. A heterozygous mutation in ATM was identified in these 3 subjects but was not directly linked to the observed phenotype. However, the attenuation of ICL sensitivity was associated with a reduced recruitment of 53BP1 during the course of ICL repair, and increased HR levels. These results further demonstrate the importance of favoring HR over NHEJ for the survival of cells challenged with ICLs.SCOPUS: ar.jinfo:eu-repo/semantics/publishe