A Two-Color Non-Muscle SERCA FRET Sensor for Diabetes Drug Discovery Using Fluorescence Lifetime Detection

Abstract

We have developed intramolecular FRET sensors capable of detecting cytoplasmic headpiece movements of human SERCA (sarco/endo-plasmic reticulum calcium ATPase) in live-cell assays, including a non-muscle SERCA2b isoform to be used in drug discovery for treatment of diabetes. Two fluorescent proteins, clover (green) and mRuby2 (red) were directly fused to selected locations on human SERCA1a (skeletal muscle), 2a (cardiac muscle), and 2b (non muscle), based on a previously reported SERCA2a construct (Gruber et al., J. Biol. Screening, 2014), and expressed stably in HEK cells. We have used these cells in a novel fluorescence lifetime plate reader (FLT-PR) to screen small-molecule libraries, to discover modulators of SERCA structure and function. The present study focuses on SERCA2b, with the goal of obtaining small molecules that activate SERCA in non-muscle cells. Since recent reports indicate that SERCA overexpression in non-muscle cells can alleviate Type II diabetes, we seek small-molecule SERCA activators for the same purpose. The small-molecule modulators identified in the high-throughput FRET screen were examined for their ability to affect SERCA's function, through assays of ATPase and calcium pumping activities. In order to obtain functional data more directly related to Type II diabetes, we tested the compound's alleviation of endoplasmic reticulum stress in 3T3-L1 adipocytes, using an XF24 Extracellular Flux Analyzer to measure mitochondrial function after inducing ER stress with the inflammatory cytokine TNF-α. While this study is designed to find activators of SERCA2b for treatment of diabetes, constructs based on other SERCA isoforms show promise in targeted therapeutics for muscular dystrophy (SERCA1a) and heart failure (SERCA2a).