HTS Calcium Flux Assay

The purities of compounds in the Vanderbilt HTS facility are more than 95% as confirmed by the supplier. A total of 35,288 compounds were tested for their ability to modulate the Y₄R activation in conjunction with the Vanderbilt HTS facility. In a pilot HTS experiment, 2000 compounds (spectrum collection, MicroSource Discovery Systems, Inc., Gaylordsville, CT) were screened for modulation of the Y₄R. This collection is designed to enrich the general hit rate by including drugs with known biological profiles (60%), naturally occurring products with no biological profile (25%), and non-drug compounds with biological profiles (15%). In a following HTS, 33,288 compounds were tested for Y₄R modulatory effects. Thirty-two thousand of these compounds were randomly selected from the Vanderbilt compound library and 1288 were selected based on their similarity to Niclosamide, a PAM discovered in the pilot HTS.

Cells were plated in TC-treated 384-well plates (black, clear bottom, Greiner, Monroe, NC) in 20 μL cell culture medium using a Multidrop Combi (Thermo Fisher, Waltham, MA) microplate dispenser (ThermoScientific, Thermo Fisher) at a density of 16,000 cells/well. The cells were incubated for 24 hours at 37°C in the presence of 5% CO₂. Following incubation, the medium was replaced with 20 μL/well fluorescent dye solution (1.0 μM Fluo-2 AM (TEFlabs, Austin, TX), .01% (v/v) Pluronic Acid F-127 in assay buffer) using an ELx405 cell washer (BioTek, Winooski, VT). Following a 90 minute incubation at room temperature, fluorescent dye solution was replaced with 20 μL/well assay buffer (HBSS, 20 mM HEPES, and 1.25 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) using the ELx405 and cell plates were loaded into a Functional Drug Screening System (FDSS, Hamamatsu, Japan). Once loaded into the FDSS, cell plates were imaged at 1Hz (excitation 470 ± 20 nm, emission 540 ± 30 nm using a 3-addition protocol designed to detect agonists, potentiators, and inhibitors: 1) after collecting 4 seconds of baseline, 20 μl/well of 20 μM test compounds in assay buffer + 0.1% (w/v) fatty-acid-free bovine serum albumin (Sigma-Aldrich, modified assay buffer) were added 2) following a 150 second delay, 10 μl/well of concentration of 5-fold over the PP EC₂₀ (55 ± 27 pM) 3) after 330 seconds, a 13 μl/well addition 5-fold over the PP EC₈₀ (836 ± 33 pM) in modified assay buffer was performed. On each screening day, PP EC₂₀ and EC₈₀ plates were adjusted after a test PP CRC at the beginning of each day to account for minor day to day variations in experimental conditions.