IMPROVED CA2+-ACTIVATED PHOTOPROTEINS FOR HTS APPLICATIONS

INTRODUCTION The Ca2+ bivalent ion is a critical second messenger involved in many physiological and signal transduction processes within the cell. Transient changes in intracellular Ca” concentrations are mainly triggered either by ligand-mediated activation of GPCRs (G-protein coupled receptors) or by opening of Ca” permeable ion channels. Both are transmembrane proteins and belong to the most preferred target classes within the pharmaceutical industry. The development of precise Ca2+ mobilization assays for HTS (High Throughput Screening) applications is challenged by the nature of the Ca” signal which is rapid, short, transient and sometimes weak.’ The use of a Ca2+ activated photoprotein with its inherent property of flash-type luminescence represents a valid tool for analyzing all aspects of Ca2+ mediated signal transduction processes in mammalian cells. The advantages of using photoproteins as Ca” indicators include: low background levels resulting in a good signal-to-noise ratio; monitoring of reaction kinetics, and possibility to measure Ca2’ concentrations at specific cellular sites. While the miniaturization of the HTS process for testing chemical compound libraries has allowed the scaling up of the number of welldplate fiom 96 to 384 and up to 1536 and a saving on the volumes of compounds used, the lower number of cells/well means that the reporter system must necessarily generate a strong and reproducible signal that can be accurately detected. To face these challenges, we are engaged in finding and developing new photoproteins with novel or improved properties, such as Ca2+ affinity, higher light release, slower kinetics.