Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET

Abstract

We have used a time-resolved FRET assay to develop a system for detecting small-molecule modulators of actin-myosin structure and function. The fluorescent donor was fluorescein attached to actin C374, and the non-fluorescent acceptor was Dabcyl attached to the N-terminus of a peptide containing the 12 N-terminal amino acids of the A1 essential light chain (DNT). FRET was measured from the decrease in fluorescence lifetime, measured by time-resolved fluorescence in a novel fluorescence plate-reader, resolving structural distributions of the bound complex. The DNT binding site on actin overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-DNT complex as in the actin-myosin complex and (b) a significant decrease in actin-DNT FRET upon binding skeletal and cardiac myosin. A high-throughput FRET screen of a small-molecule library (NCC) showed that actin-DNT FRET is significantly affected by some compounds. All of these compounds inhibit actin-activated skeletal and cardiac myosin ATPase and affect the microsecond dynamics of actin, as detected by transient phosphorescence anisotropy. We concluded that the actin-DNT system allows detection of the effects of pharmacologically active compounds on actin structural states and consequently on functional properties of actomyosin. These results set the stage for a drug-discovery campaign, seeking therapies for cardiomyopathy and other disorders of involving actin-myosin dysfunction.