title: High-throughput FRET assay yields allosteric SERCA activators.
abstract: Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gruber
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Lockamy
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.forename: Dongzhu
authors.initials: D
authors.lastname: Jin
authors.forename: Jiqiu
authors.initials: J
authors.lastname: Chen
authors.forename: Russell
authors.initials: R
authors.lastname: Dahl
authors.forename: Tamas
authors.initials: T
authors.lastname: Bartfai
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Zsebo
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Gillispie
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002135:Calcium-Binding Proteins / Q000502:physiology; D002478:Cells, Cultured; D020536:Enzyme Activators / Q000494:pharmacology*; D057075:Enzyme Assays; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D013268:Stimulation, Chemical; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D020536:Enzyme Activators; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban