A Large-Scale High-Throughput Screen for Modulators of SERCA Activity

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Abstract

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.

Original languageEnglish (US)
Article number1789
JournalBiomolecules
Volume12
Issue number12
DOIs
StatePublished - Dec 2022

Bibliographical note

Funding Information:
This work was supported by NIH grants R01HL139065 (formerly GM027906) to D.D.T. and R.L.C., R37AG026160 to D.D.T., and AHA grant 20CDA35310575 to PAB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2022 by the authors.

Keywords

  • calcium ATPase
  • calcium transport
  • cardiac muscle
  • drug discovery
  • heart failure
  • membrane transport

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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