Category: Assay Development and Screening
The measurement of Ca2+ flux is routinely monitored using an intracellular dye that fluoresces when bound to Ca2+. We developed an HTS-compatible microsome-based assay on the FLIPR Tetra which, unlike classical cell-based FLIPR assays, monitors changes in free Ca2+ levels in the buffer as Ca2+ is transported into the vesicle via an endogenous Ca2+-ATPase. The assay utilizes microsomes isolated from native tissue which are treated with ATP and then Ca2+ flux into the microsomes is measured as a decrease in fluorescence. The Ca2+ concentration in the buffer is controlled through chelation with EGTA, as is standard practice in Ca2+ sensitive assays. In the course of the screening campaign, some unusual SAR trends emerged which lead to a forensic analysis of the source. We will present our data that identified unappreciated metal contamination in several compounds that lead to a change in the Ca2+-EGTA equilibrium and resulted in a false positive read in the assay. We will detail our strategy for identifying false positives that includes monitoring the FLIPR baseline fluorescence and analyzing compounds for metal contaminants using Icagen’s XRpro® technology. These efforts were implemented to mitigate against unnecessary follow-up.
Ingrid Stock– principal scientist, Pfizer, Groton, Connecticut