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

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.

Keywords: calcium ATPase; calcium transport; cardiac muscle; drug discovery; heart failure; membrane transport.

Figure 1

DIVERSet ATPase screen for SERCA effectors. (A) Percent change in ATPase activity for 46K compound screen. Selected activating compounds displayed in red. (B) Gaussian fit of percent effect distribution. (C) CV% for each assay plate. (D) Numbers of SERCA activating and inhibiting compounds that meet various SD thresholds above (activators) or below (inhibitors) the mean.

Figure 2

Representative CRC of SERCA-activating compound #12 for ATPase activity assay (% change in activity relative DMSO control) performed at pCa 5.4 in skeletal SR (V_max).

Figure 3

Representative chemical structures from each cluster set.

Figure 4

Linked Activating Compounds: Concentration response curves of compounds #9 (DS84875791) in cardiac SR and #7 (DS31221514) display a comparable change at lower compound concentrations, although ATPase achieves a greater maximal effect at the highest compound concentrations. n = 3, ± SEM.

Figure 5

Improved Coupling Compounds: Concentration response curves of compounds #3 (DS76145530) in cardiac SR and #8 (DS39746187) in skeletal SR show a percent increase in Ca²⁺ uptake activity that precedes the ATPase effect indicating improved coupling efficiency between the two SERCA enzymatic activities. n = 3, ±SEM.

Figure 6

Uncoupling Compounds: Concentration response curve of compound #16 (DS33804556) in cardiac SR and skeletal SR show an inhibition of Ca²⁺ uptake activity despite a substantial increase in ATPase activity.

Figure 7

Isoform-specific uncoupler. Concentration response curve of compound #12 (DS60405307) shows an inhibition of Ca²⁺ uptake activity despite a substantial increase in ATPase activity in cardiac SR. However, the same compound has a small but significant increase in Ca²⁺ uptake activity in skeletal SR. This tissue-dependent effect is presumably due to SERCA2a (cardiac) vs. SERCA1a (skeletal) isoform differences or the presence/absence of regulators (PLB vs. SLN). n = 3, ± SEM.