Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MS 55455, USA.
Cells. 2020 May 8;9(5):1170. doi: 10.3390/cells9051170.
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.
我们设计了一种串联荧光生物传感器和双波长荧光寿命（FLT）检测方法，以在活细胞中进行高通量筛选（HTS），从而发现潜在的心力衰竭药物。心力衰竭与肌浆网 Ca-泵（SERCA2a）的活性不足有关，这通常是由于磷蛋白（PLB）过度抑制所致，PLB 是一种小的跨膜蛋白。我们试图发现通过破坏 PLB 和 SERCA2a 之间的这种抑制性相互作用来恢复 SERCA2a 活性的小分子。我们的方法是荧光标记这两种蛋白质，并测量荧光共振能量转移（FRET）以检测复合物结合或结构的变化。为了优化对这些变化的敏感性，我们设计了一种生物传感器，将两个荧光标记的蛋白质串联在单个多肽链上。这种 SERCA2a-PLB FRET 生物传感器构建体具有功能活性，可有效用于 HTS。通过在初级 HTS 期间以极高的速度实施 2 波长 FLT 检测，我们将荧光化合物作为假阳性 Hits 剔除。在试点筛选中，我们鉴定了改变 SERCA2a-PLB 相互作用的 Hits，并且新开发的二次钙摄取测定法揭示了 Ca 转运的激活剂和抑制剂。我们正在实施这种方法进行大规模筛选，以发现用于心力衰竭治疗开发的新的类似药物的 SERCA2a-PLB 相互作用调节剂。
