Downstream Regulatory Element Antagonist Modulator (DREAM) is a neuronal calcium sensor protein which is highly expressed in several areas of the central nervous system such as the hippocampus and the neural cortex. It has been related to several physiological processes and linked to pathological conditions such as Alzheimer’s disease. DREAM interacts with multiple intracellular partners and has a significant role in regulating gene expression, kinetics of potassium channels, calcium homeostasis, and enzymatic activity of presenilin. Previous studies have shown that the insulinotropic agent repaglinide targets neuronal calcium sensors by selectively binding to them in a calcium dependent manner. It has also been shown that DREAM binds to small hydrophobic molecules such as 1,8-ANS, CL 88, and NS5806. Here we aim to test the role of repaglinide as a neuronal calcium sensor antagonist through characterizing its interactions with DREAM and monitoring its impact on protein structure and dynamics. Fluorescence studies showed a decrease in the Trp 169 emission upon repaglinide addition, providing a Kd value of 30 mM in the presence of Ca2+and similar Kd value was determined for the apoprotein. Association of repaglinide to DREAM leads to a decrease in the Trp169 lifetime suggesting a dynamic quenching mechanism. Titration of DREAM-1,8-ANS complex with repaglinide resulted in a decrease in the emission intensity, pointing towards the binding of repaglinide to one of the 1,8-ANS binding sites. Using AutoDock and molecular dynamics we show that repaglinide binds to a hydrophobic cavity located between the N- and C- terminal and the DREAM-repaglinide complex is stabilized through hydrogen bonds with Lys166 and Arg160. Those results suggest that several members of NCS protein family may serve as target for repaglinide.

Neuronal calcium sensor DREAM interactions with insulinotropic agent repaglinide

2019 Spring National Meeting

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