08.05.2018 | Webinar: HTS Sodium Ion Channel Assays on the SyncroPatch 384PE
SyncroPatch 384PE (a predecessor model of SyncroPatch 384i)
Learn about Sodium Ion Channel Assays on the SyncroPatch 384PE. Our speakers will present assays on NaV1.1, NaV1.7, NaV1.8 and NaV1.9:
- Biophysical and Pharmacological Characterization of Voltage-Gated Na Channels Involved in Pain Pathways.
- Identifying NaV1.1 Enhancers to Restore Inhibitory Interneuron Function in Dravet Syndrome and Alzheimer’s Disease.
Gesa Rascher-Eggstein, Nanion Technologies.
Markus Rapedius, Nanion Technologies.
Biophysical and Pharmacological Characterization of Voltage-Gated Na Channels Involved in Pain Pathways.
Voltage-gated Na (NaV) channels expressed in dorsal root ganglion neurons (DRG) such as Nav1.7, NaV1.8 and NaV1.9 have been proposed to play important roles in nociception and pain signalling1. Besides NaV1.7, NaV1.8 and NaV1.9 are exclusively expressed in dorsal root ganglion (DRG) neurons where they have been associated in neuropathic and inflammatory pain1 or linked to inherited pain syndromes2. Whereas NaV1.7 plays a pivotal role in the modulation of action potential threshold, NaV1.8 channel is the predominant channel driving and shaping TTX-resistant action potentials (AP) in DRG neurons. Due to its relatively depolarized voltage dependence of inactivation, NaV1.8 can contribute to action potential generation even at depolarized membrane potentials which may occur during nerve injury or pain signalling3. This property, coupled with its location in DRG neurons and the modification of expression patterns in animal models of pain and human pain states, has meant that NaV1.8 has received attention as a novel target for pain therapeutics for chronic, inflammatory and neuropathic pain. Although NaV1.9 probably does not contribute to action potential amplitude, it most likely acts as a threshold channel, contributing to resting membrane potential and lowering the threshold for action potentials thereby increasing repetitive firing4. Gain-of function mutations in human pain disorders points to a role of NaV1.9 in pain sensation and transmission in humans. However, NaV1.7 is TTX-resistant and does exhibit distinguished biophysical characteristics such as fast inactivation and slow recovery from inactivation.
We have used automated patch clamp in combination with commercially available cell lines to investigate the activation and inactivation properties of NaV1.7, NaV1.8 and NaV1.9 in comparison. Furthermore, we have addressed pharmacological properties of different inactivated states of standard compounds. We are happy to provide examples for all three channels at high success rate ready for Drug Discovery.
1 Amaya, et al, 2000. Mol & Cell. Neurosci. 15:331-42
2 Theile JW and Cummins TR., FrontPharm; 2:54; 2011.
3 Clare, 2010. Expert Opin. Investig. Drugs. 19(1):45-62
4 Dib-Hajj, et al, 2015. Nat. Rev. Neurosci. 16(9): 511-519
Elisa Ballini, Evotec AG.
Identifying Nav1.1 Enhancers to Restore Inhibitory Interneuron Function in Dravet Syndrome and Alzheimer’s Disease.
Elisa's work was presented at the BPS 2018 as poster.
Further authors of the poster:
Elisa Ballini1, Ben Liu2, Armando Gutierrez2, Daniela Borchert1, Sandrine Saillet2, Ann Kathrin Niehaus1, Bernhard Freigassner1, Petra Friess1, Stephen Hess1, Lennart Mucke2,3, Jorge Palop2,3
1Evotec AG, Hamburg, Germany
2Gladstone Institute of Neurological Disease, San Francisco, CA 94158, USA
3Cure Network Dolby Acceleration Partners, LLC., San Francisco, CA 94158, USA
Brain dysrhythmias in diverse conditions, including Alzheimer’s disease (AD) and Dravet syndrome (DS), are associated with hypofunction of the voltage-gated sodium channel subunit NaV1.1, which is predominantly expressed in inhibitory interneurons. NaV1.1 enhances interneuron function and promotes brain rhythms (e.g., gamma oscillations) that are required for cognitive function and disrupted in these disorders. In animal models of AD, enhancing gamma oscillations by increasing NaV1.1 levels or by optogentic stimulation of NaV1.1-expressing interneurons improved cognitive function and reduced amyloid pathology.
We aim to develop small-molecule drugs that selectively increase the activity of NaV1.1 (“NaV1.1 enhancers”) to restore inhibitory interneuron functions and augment gamma oscillations in DS, AD, and related conditions.
We completed a 50,000 small-molecule high-throughput screening (SyncroPatch platform) on cells expressing human NaV1.1. We identified enhancers of NaV1.1 activity and performed hit confirmation studies, dose-response curves, chemical quality control and medicinal chemistry analyses on selected hits. We developed interneuron- and NaV1.1-dependent secondary functional assays for gamma oscillations in brain slices and freely behaving mice for ex vivo and in vivo validation and efficacy studies. We confirmed several hits that consistently enhance Nav1.1 currents in recombinant cells and interneuron-dependent gamma oscillations in brain slices. Analogs of the identified hits are being tested for structure-activity relationships (SAR) and Nav subunit selectivity. Mouse models of DS and AD have been selected as the preferred disease models to assess therapeutic efficacy of these and related compounds. Conclusion: Our high-throughput screening successfully identified novel small molecules that enhance NaV1.1 function and interneuron-dependent gamma oscillatory activity in complex circuit and may be suitable for full lead optimization.
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