Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules that Enhance Ca2+ Transport

Cells

Evan Kleinboehl¹  AndrewR. Thompson¹  RazvanL. Cornea¹  DavidD. Thomas¹  LaurenN. Roelike¹  ToryM. Schaaf¹  SamanthaL. Yuen¹  Bengt Svensson¹ 

[1] Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MS 55455, USA;

关键词: time-resolved FRET;    biosensor;    drug screening;    SERCA2a;    small-molecule;    HTS;   

DOI  :  10.3390/cells9051170

来源: DOAJ

【 摘 要 】

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

【 授权许可】

Unknown