Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit.Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

Voltage-gated Na+ (NaV) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial NaV channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial NaV remains largely unexplored. Here we systematically screened 39 NaV modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human NaV1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both NaV1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases NaV1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of NaV1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.

Ion channels regulate a variety of physiological processes and represent the second largest class of known drug targets. Among the known methods to study ion channels, patch clamp electrophysiology and its automated versions, automated patch clamp (APC), have become the gold standard with unsurpassed precision for ion channel functional assays. However, due to the complexity of electrophysiological data, the lack of robust standard data analysis methods became the major bottleneck for high-throughput APC application in ion channel drug discovery. This poster illustrates how a collaboration between Genentech and Genedata has extended the boundaries of kinetic data analysis throughput and complexity. The elements of this achievement are: 1) A data parser for raw instrument recordings, allowing for near-instantaneous data analysis upon data capture, including voltage-current data from the Nanion SyncroPatch 768PE instrument, 2) A high-performing implementation of Boltzmann fitting models to infer half-activation voltage and half-inactivation voltage shifts between pre- and post-compound conditions, 3) Embedding of a workflow providing visual and statistical quality control of result quality on various levels, considering the four key parameters seal resistance, peak current, capacitance, and series resistance. The analysis is tailored to real-life situations, e.g., parallel assessment of multiple mutant/WT channels, and one-click downstream-reporting of data. In summary, the fast advancing APC high-throughput technologies together with the newly-developed robust and automated high-throughput data analysis methods will have a significant impact on ion channel study and drug discovery.

Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high-throughput electrophysiology and its applications in drug discovery is provided. © 2019 The Authors.

Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC)-derived cortical neurons (iCell Neurons and iCell GlutaNeurons). We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents.

To explore a method that can prevent haematopoietic cells from chemotherapeutic drugs during chemotherapy of tumor. MDR1 gene was transfered to cord blood nucleate cells (CBNC) mediated by adenovirus vector, for enhancing its resistance to chemotherapeutic drugs. The CBNC transferred with MDR1 gene have significant resistance to chemotherapeutic drugs compared with the CBNC without gene transfer (P0. 01). The CBNC transferred with MDR1 gene can obviously resist the damage of chemotherapeutic drugs, provide a method to prevent the cells damage during chemotherapy of patients, and enhance the effect of chemotherapy.