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Coupling automated electrophysiology with microfluidic perfusion to drive hit to lead and lead optimization studies for voltage- and ligand-gated ion channel targets. (W528)
Clark G, Hollands E, Shearer J, Southan A.
Rapid and highly accurate profiling of compound activity is essential for the timely progression of drug discovery research; especially during hit-to-lead and lead optimisation cycles, where inaccurate reporting of compound activity can result in the project team making poor strategic decisions. To investigate a novel option to evaluate compounds against ligand-gated ion channels we have generated a validation dataset for a GABAA ion channel stably expressed in HEK-293 cells using a Dynaflow® HT automated patch-clamp system. In a separate study we also examined the voltage-gated potassium channel Kv1.5 expressed in a CHO-K1 background. The instrument is based upon a 96 well recording consumable containing microfluidic channels and uses laminar flow to separate solution streams. This laminar flow, which is decoupled from the instruments pipettor-based liquid handling, facilitates rapid switching of solutions producing 10-90% exchange times around 30 ms. For all experiments presented below the extracellular recording solution was Dulbecco’s phosphate buffered saline, the internal recording solution (in mM): 145 KCl, 3.5 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA and the holding potential -60 mV (GABAA) or -80 mV (Kv1.5).
Initial experiments focused upon sealing performance of the GABAA cell line, with a total of five recording plates examined over separate days. The percentage of wells sealing to > 30 MΩ was 71%, 69%, 81%, 68% and 84% on each of the days; with mean seal resistance ranging between 70.8 ± 24.2 to 97.6 ± 37.3 MΩ (mean ± SD; n=81 & 68 cells). Mean GABA-evoked current amplitude was consistent throughout, with day one mean amplitude being 1.36 ± 0.88 nA (n=22 cells) and the day five mean amplitude being 1.58 ± 0.9 nA (n=55 cells). The GABA EC50 was consistent over three separate days at 2.2 µM (slope 1.5), 2.5 µM (slope 1.3) and 3.0 µM (slope 1.4) [n= 4 - 13 recordings per concentration point, 300 ms GABA application]. On two separate occasions the GABAA blocker bicuculline was also examined producing IC50 values of 1.1 µM and 1.4 µM.
Kv1.5 currents were evoked by a one second step to +40 mV, with a ten second inter-pulse interval. In a six plate fully automated experiment mean seal resistance ranged between 92.1 ± 36.2 MΩ to 104.0 ± 40.4 MΩ and mean Kv1.5 current amplitude ranged between 1.8 ± 0.9 nA to 2.4 ±1.1 nA (n=64 to 86). Capsaicin and 4-AP pharmacology was also examined; IC50 values were 203 µM (slope 0.6) and 30 µM (slope 0.6) respectively (n= >15 recordings per concentration point).
We will present recording performance statistics and pharmacological data to support the use of the Dynaflow® HT assay platform in drug discovery research.
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