Voltage Sensor Probes (VSPs) as an Efficient Tool to Screen for Inhibitors of Voltage-Gated Sodium Channels

Voltage-gated sodium channels (Nav) represent a therapeutically validated group of targets for the development of antiepileptic drugs, analgesics and antiarrhythmics [1]. However most of the existing drugs acting as Nav blockers suffer from multiple side effects, but the existence of a multigene family of Nav [2] suggests that the identification of new compounds that selectively block Nav isoforms might have better therapeutic efficiency and reduced side effects. Due to their molecular interference with numerous ion channels, alkaloids represent a group of natural products of particular interest. This is the reason why we have evaluated the efficiency of an in-house method to screen a library of isoquinoline alkaloids formerly isolated in our laboratory. Mammalian GH3 cells constitutively expressing Nav where used in conjunction with Voltage Sensor Probes (VSPs), the signals being read on a fluorescence plate reader. Thanks to this technique, we were able to precisely detect Nav channels activators or blockers. Among 62 compounds tested, 5 isoquinolines appeared as potent Nav channels inhibitors. References: 1. Salat, K. et al. (2014) EOID 23:1093-1104 2. Yu, F.H. et al (2003) Genome Biol. 4

Screening an in house alkaloids library using Voltage-Sensor Probes for new modulators of voltage-gated sodium channels

Voltage-gated sodium channels (Nav) are molecular targets of clinically used drugs for treatments of various diseases (epilepsy, chronic pain, cardiac arrhythmia…) and also of numerous animal and plant neurotoxins. The development of easy-to-use screening assays for searching new ligands from chemicals libraries, animal venoms or plant extracts represents a challenge of a great interest to generate therapeutic hits. Here, we used the mammalian GH3B6 pituitary cell line, which constitutively expresses three different neuronal Nav channel isoforms (Nav1.2, Nav1.3 and Nav1.6), to identify novel compounds of pharmacological interest from a library of in-house vegetal alkaloids. The screening is based on a method using Voltage-Sensor Probes (VSPs) that we adapted to detect both activators and blockers of Nav channels. Over the 84 pure alkaloids or plant extracts that were screened, 17 increased the VSP signal. They operated as gating modifier, showing an action mechanism similar to that of batrachotoxin (BTX), known to strongly inhibit Nav channel inactivation. The remaining 67 plant products were assessed for their potency to inhibit BTX-induced VSP signal. We further selected 11 alkaloids as efficient Nav channels inhibitors. We focused our attention on two structural analogs belonging to the aporphine family, liriodenine and oxostephanine, which differ only by a methoxy group. Whereas liriodenine has been already described as a Nav channels blocker, oxostephanine has not been yet documented as an ion channel modulator. In conclusion, the novel VSPs-based screening assay we developed is a suitable method to challenge the discovery and to assess the activity of novel ligands on Nav channels

The Wide Brown Dwarf Binary Oph 1622-2405 and Discovery of A Wide, Low Mass Binary in Ophiuchus (Oph 1623-2402): A New Class of Young Evaporating Wide Binaries?

We imaged five objects near the star forming clouds of Ophiuchus with the Keck Laser Guide Star AO system. We resolved Allers et al. (2006)'s #11 (Oph 16222-2405) and #16 (Oph 16233-2402) into binary systems. The #11 object is resolved into a 243 AU binary, the widest known for a very low mass (VLM) binary. The binary nature of #11 was discovered first by Allers (2005) and independently here during which we obtained the first spatially resolved R~2000 near-infrared (J & K) spectra, mid-IR photometry, and orbital motion estimates. We estimate for 11A and 11B gravities (log(g)>3.75), ages (5+/-2 Myr), luminosities (log(L/Lsun)=-2.77+/-0.10 and -2.96+/-0.10), and temperatures (Teff=2375+/-175 and 2175+/-175 K). We find self-consistent DUSTY evolutionary model (Chabrier et al. 2000) masses of 17+4-5 MJup and 14+6-5 MJup, for 11A and 11B respectively. Our masses are higher than those previously reported (13-15 MJup and 7-8 MJup) by Jayawardhana & Ivanov (2006b). Hence, we find the system is unlikely a ``planetary mass binary'', (in agreement with Luhman et al. 2007) but it has the second lowest mass and lowest binding energy of any known binary. Oph #11 and Oph #16 belong to a newly recognized population of wide (>100 AU), young (<10 Myr), roughly equal mass, VLM stellar and brown dwarf binaries. We deduce that ~6+/-3% of young (<10 Myr) VLM objects are in such wide systems. However, only 0.3+/-0.1% of old field VLM objects are found in such wide systems. Thus, young, wide, VLM binary populations may be evaporating, due to stellar encounters in their natal clusters, leading to a field population depleted in wide VLM systems.Comment: Accepted version V2. Now 13 pages longer (45 total) due to a new discussion of the stability of the wide brown dwarf binary population, new summary Figure 17 now included, Astrophysical Journal 2007 in pres

Voltage Sensor Probes as an efficient tool to screen for new modulators of voltage-gated sodium channels

Voltage-gated sodium channels (Nav) constitute the molecular targets of clinically used drugs for treatments of various diseases (epilepsies, chronic pain, cardiac arrythmies…) and also of numerous toxins from animals and plants. The development of useful screening assay for the discovery of new ligands in/ from chemicals libraries or animal venoms and either plant extract still represents a challenge of great interest. Here, we used a mammalian GH3 cells that constitutively express at least three different brain Nav channels isoforms (Nav1.1, Nav1.2, Nav1.3 and Nav1.6) in order to identify in a library of in-house natural alkaloids, novel compounds of therapeutical interest. For this screening, we developed a method based on the use of Voltage Sensor Probes (VSPs) that we adapted to detect both activators and blockers of Nav channels. Among 62 compounds tested, 5 isoquinolines appeared as potent Nav channels inhibitors. Other compounds were characterized as specific gating modifier of Nav channels. While most of these alkaloids have been already described in the literature, their abilities to act on Nav channels were unknown. In conclusion, we demonstrated the potency of this novel screening method using VSPs to identify novel ligands of Nav channels of therapeutical interests. References: 1. Salat, K. et al. (2014) EOID 23:1093-1104 2. Yu, F.H. et al (2003) Genome Biol. 4

High accuracy 234U(n,f) cross section in the resonance energy region

New results are presented of the 234U neutron-induced fission cross section, obtained with high accuracy in the resonance region by means of two methods using the 235U(n,f) as reference. The recent evaluation of the 235U(n,f) obtained with SAMMY by L. C. Leal et al. (these Proceedings), based on previous n-TOF data [1], has been used to calculate the 234U(n,f) cross section through the 234U/235U ratio, being here compared with the results obtained by using the n-TOF neutron flux

Characterization of the n-TOF EAR-2 neutron beam

The experimental area 2 (EAR-2) at CERNs neutron time-of-flight facility (n-TOF), which is operational since 2014, is designed and built as a short-distance complement to the experimental area 1 (EAR-1). The Parallel Plate Avalanche Counter (PPAC) monitor experiment was performed to characterize the beam prole and the shape of the neutron 'ux at EAR-2. The prompt γ-flash which is used for calibrating the time-of-flight at EAR-1 is not seen by PPAC at EAR-2, shedding light on the physical origin of this γ-flash