External ID: VANDERBILT_HTS_GIRK2_HPP

Protocol: To screen for modulators of the G protein-gated inwardly-rectifying potassium channel subunit 2 (GIRK2) homomeric channels, HEK293 cells were engineered to overexpress GIRK2 and the neuropeptide Y receptor type 4 (NPY4). The purpose of NPY4 overexpression was to promote modest activation (~30% activity) of GIRK2 channels upon NPY4 activation with human pancreatic polypeptide (hPP). These cells were screened using a high-throughput thallium-flux assay in a 384-well format. High-throughput thallium flux assays are based on three principles: (1) extracellular thallium can permeate through potassium channels into cells, (2) the fluorescent dye Thallos enters and stays in the cytoplasm of HEK293 cells, and (3) the fluorescence of Thallos increases dramatically when it interacts with thallium ions. Compounds that modulate the activity of an overexpressed channel, in this case GIRK2, are identified by the change in fluorescence that results from changes in thallium influx into cells through the overexpressed channel.

The screening protocol was conducted as follows. One hour prior to screening, cells were loaded with 1.5 micromolar (muM) Thallos in assay buffer (1x Hank's Balanced Salt Solution and 20 millimolar (mM) HEPES). The liquid handling and imaging was conducted using a kinetic-imaging plate reader, Panoptic (WaveFront Biosciences). Before screening, Thallos-containing buffer was removed from cells and replaced with 20 microliters (muL) per well of assay buffer. Compounds were dissolved in assay buffer at 20 muM. After 8 seconds of imaging, 20 muL of compound solution was added per well and allowed to incubate for 150 seconds. Next, 10 muL of a solution containing 2.0 mM thallium and 3.5 nanomolar (nM) hPP in Base Buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5mM Glucose, and 20 mM HEPES) was added to the wells. 30 seconds later, 12 muL of base buffer containing 2.0 mM thallium and 1 muM hPP, a maximally-activating concentration, was added to each well. Data was collected for 60 seconds after this final addition. The positive control for this screen was 16.6 muM eprinomectin, a natural product previously identified as a GIRK2 activator as part of a small-scale pilot screen. Microsoft Excel was utilized for data analysis. Data were normalized, F/Fo, on a well-to-well basis to account for differences in cell number. Compound activity was analyzed by comparing the sums of control-subtracted amplitudes of fluorescence intensity at 20 seconds after each thallium addition. The top 0.5% compounds corresponding to wells that demonstrated the largest increases in fluorescence were selected as "hits" for counter screening, as described below.

All 34,131 compounds screened using the method described above are listed in column OUTCOME_SCREEN_HITS. The 167 compounds selected as hits based on the above criteria are labelled ACTIVE in this column. Fluorescent compounds were included in the inactive compounds and marked as INACTIVE.

The 167 active compounds from column OUTCOME_SCREEN_HITS were tested for non-specific activity in untransfected HEK293 cells using assay conditions otherwise identical to the primary screen. Column OUTCOME_HEK_COUNTERSCREEN lists the 39 compounds that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 128 compounds which were identified as hits in the primary screen but inactive when tested in untransfected HEK293 cells were tested for activity in GIRK2-overexpressing HEK293 cells that did not express NPY4 receptor. Column OUTCOME_GIRK2_COUNTERSCREEN lists the 8 compound that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 8 active compounds from column OUTCOME_GIRK2_COUNTERSCREEN were tested at various concentrations to determine if compound activity was concentration dependent. Column OUTCOME_GIRK2_DOSE_RESPONSE provides the efficacy of these compounds; activity of all compounds at the provided concentrations was normalized to the activity of VU0529331 at 37.5 muM, which was the highest efficacy observed during this screening. For each compound concentration listed, the efficacy of that compound provided refers to the normalized, control-subtracted fluorescence intensity at 15 seconds after the addition of thallium. Compounds that demonstrated >5% efficacy and concentration-dependent activity were labelled as ACTIVE.

Finally, column Outcome combines all of the information from the above counterscreens together, indicating the 6 hits from this GIRK2 screen.

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