A 1,536-well ultra-high-throughput siRNA screen to identify regulators of the Wnt/beta-catenin pathway.

High-throughput siRNA screens are now widely used for identifying novel drug targets and mapping disease pathways. Despite their popularity, there remain challenges related to data variability, primarily due to measurement errors, biological variance, uneven transfection efficiency, the efficacy of siRNA sequences, or off-target effects, and consequent high false discovery rates. Data variability can be reduced if siRNA screens are performed in replicate. Running a large-scale siRNA screen in replicate is difficult, however, because of the technical challenges related to automating complicated steps of siRNA transfection, often with multiplexed assay readouts, and controlling environmental humidity during long incubation periods. Small-molecule screens have greatly benefited in the past decade from assay miniaturization to high-density plates such that 1,536-well nanoplate screenings are now a routine process, allowing fast, efficient, and affordable operations without compromising underlying biology or important assay characteristics. Here, we describe the development of a 1,536-well nanoplate siRNA transfection protocol that utilizes the instruments commonly found in small-molecule high throughput screening laboratories. This protocol was then successfully demonstrated in a triplicate large-scale siRNA screen for the identification of regulators of the Wnt/beta-catenin pathway.

A high-throughput assay for evaluating state dependence and subtype selectivity of Cav2 calcium channel inhibitors.

Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.

A cell-sparing electric field stimulation technique for high-throughput screening of voltage-gated ion channels.

The Trans Cell Layer Electrical Field Stimulation (TCL-EFS) system has been developed for high-throughput screening (HTS) of voltage-gated ion channels in microplate format on a Voltage-Ion Probe Reader (VIPR) platform. In this design, a wire electrode is placed above the cell layer of each filter well, and a whole plate perimeter electrode resides beneath the filter layer. This configuration allows the electrodes to be placed away from the cell layer to minimize the near electrode field effects on cell function and dye bleaching observed with other existing designs. Mathematical simulation indicates that the electric field at the cell layer becomes uniform as the top electrode is raised to a position near the surface of the solution in the well. Using the TCL-EFS system and membrane potential fluorescence resonance energy transfer (FRET) dyes, the sensitivity of voltage-gated sodium channels to tetrodotoxin and other channel inhibitors was found to be similar to those determined by established electrophysiological and more conventional VIPR techniques. A good correlation was also observed with the TCL-EFS system for inhibition of Cav2.2 by omega-conotoxin-GVIA and for block of Cav1.2 by known small molecule inhibitors. Thus, the TCLEFS system is suitable for both quantitative analysis and HTS of voltage-gated sodium and calcium channels, without the liabilities of previously reported EFS methodologies.