Morten Sunesen

Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study.

Background There is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds. Methodology/Principal Findings A comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0. Conclusions/Significance The correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC50; therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.

Automated Patch-Clamp Technique: Increased Throughput in Functional Characterization and in Pharmacological Screening of Small-Conductance Ca2+ Release-Activated Ca2+ Channels

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release—activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (I CRAC) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 ± 226 MΩ (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. ICRAC whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when ICRAC was activated by passive store depletion using EGTA. Apparent rundown of ICRAC was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. ICRAC was blocked by SKF-96365 and 2-APB with IC50 values of 4.7 ± 1.1 µM (n = 9) and 7.5 ± 0.7 (n = 9) µM, respectively. The potencies of these blockers were similar to values reported for ICRAC in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties. (Journal of Biomolecular Screening 2008:638-647)

Study of TRP Channels by Automated Patch Clamp Systems

Ion channels are responsible for the permeation of ions across the membrane and their central role in cellular physiology is well established. Historically, the direct study of ion channels has been considered technically challenging. As such, a significant barrier to drug discovery for ion channels has been the low throughput of high quality electrophysiological data. The emergence of automated high throughput platforms for studying ion channel kinetics and pharmacology has lowered this barrier. Ion channels are now recognized as increasingly important drug targets and a diverse range of ion channels are implicated in a variety of drug discovery and cardiac safety assessment programs. The TRP (Transient Receptor Potential) superfamily of ion channels play a crucial role in a broad range of sensory functions including vision, taste, olfaction, hearing, touch, pain and thermosensation. Many of the TRP channels are polymodal in their activation and deactivation mechanisms and even with conventional patch clamp electrophysiology, the TRP channels are considered to be a very complex target class. Here we present an update on the significant progress made on the TRP receptor assays with the available automated patch clamp systems.

Qube - High throughput Screening with Genuine Electrophysiology

A challenge in ion channel drug discovery is the need to screen a large number of compounds and to measure the pharmacology of drug-ion channel interaction. Automated patch clamp (APC) makes it possible to measure the pharmacology of many compounds; however, the throughput required for high throughput screening of compound libraries has been out of reach of the first generation of APC instruments. Screening of large compound libraries has been done using indirect methodologies with higher false positive and false negative errors. The Qube is a 384 channel, gigaohm-seal based APC instrument for recordings from voltage-gated and ligand-gated ion channels. It offers the capability to screen large compound libraries for ion channel block or modulation. Data are obtained with a throughput of more than 30,000 wells tested per 24 hours. In this study, we did high throughput, voltage clamp recordings of Nav1.7, hERG and ASIC1A on the Qube. Recordings were made on the QChip384 planar patch clamp consumable. Here we demonstrate throughput of up to 1500 wells tested per hour with a 95% success rate using multihole QChips. Using Nav1.7 and hERG expressing cells we demonstrate that the recording are stable and have good voltage control for at least 30 minutes. By repetitive alternating additions of high and low pH on the ASIC1A expressing cells, we demonstrate the advantages of the liquid flow system in the QChip: the QChip384 has flow channels and complete wash in/washout and indefinite waste. The capability of the Qube to handle 384 sites simultaneously combined with the QChip384 architecture enable high throughput screening of compounds with the highest output of true, direct electrophysiological recordings with an uncompromised data quality.

TRP'Ing on QPatch in Multi-Hole Mode

Transient receptor potential (TRP) channels are non-selective cationic channels that are widely distributed in mammalian tissues. Their specific physiological functions are largely unknown. Proposed functions include responses to pain, temperature, touch, osmolarity, pheromones, and taste. But due to the lack of specific blockers and the full understanding of their mechanisms of activation studies of TRP channels have been difficult and unexpectedly slow.The emergence of automated patch clamp (APC) systems has increased the number of new targets available for ion channel drug development and has augmented throughput.In order to facilitate tests of large compounds libraries on e.g. TRP targets, we have recently developed two multi-hole APC systems: QPatch HTX and QPatch 16X. The multi-hole technology allows the simultaneous recording of 10 cells in parallel per recording site thereby increasing the signal to noise ratio and the success rate.In this study, we have validated several TRP channels for their activation by their appropriate agonist e.g. Menthol, Capsaicin and temperature. We have found that using the QPatch in multi-hole mode significantly increased the volume of electrophysiology data that can be generated. Our results demonstrate that the QPatch multi-hole systems are capable of generating high quality data from a wide range of the channels belonging to the TRP family of receptors.We believe that the combination of high throughput and high quality data in a single system has more than a “transient potential” to advance the understanding of the complex mechanisms of action exhibited by difficult targets such as the TRP channels.