High-throughput FRET assay yields allosteric SERCA activators

Abstract

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

Figure 1. FRET assay for disruption of the inhibitory SERCA-PLB interaction

Top: schematic diagram illustrating possible effects of compounds on the SERCA-PLB complex. Bottom: fluorescence intensity spectra that would result (solid curve is donor-only control, F_D, dashed curve is donor-acceptor complex, F_DA). FRET efficiency is given by E = 1 − F_DA/F_D. Center: Most compounds have no effect, so the SERCA-bound donor remains quenched by FRET to the PLB-bound acceptor, giving high E value (no hit). Right: a compound that completely dissociates the complex would completely eliminate FRET, giving E = 0 (strong hit). Left: a compound that causes a structural rearrangement within the SERCA-PLB complex would cause a more subtle change in FRET.

Figure 2. Functional effects of a hit

Desirable compounds may act on the Ca²⁺ dependence of SERCA’s ATPase activity (black) to increase its apparent Ca²⁺ affinity (pK_Ca, blue) or to increase the maximum rate (V_max, red), or a combination of both (not shown). Curves represent fits of Eq. (3) to typical Ca-ATPase data.

Figure 3. High-throughput screen results

Compounds were screened, in duplicate, at a concentration of 10 μM (in DMSO). (A) Assay quality (z′) depends on the signal window (change in FRET efficiency, ΔE), and CV according to Eq. (4). (B) Compound effects on ΔE show a distribution that is fitted by a Gaussian (C), giving the average effect, μ = 0.5±0.3%, and the global SD, σ = 8%. (D) Distribution of hits.

Figure 4. ATPase assays

ATPase activity was measured after 20 min incubation in the presence of either CDN1001 (up-triangles) or CDN1033 (down triangles) or DMSO control (squares). Top (A,B): Ca-dependence in the presence and absence of 10 μM compound. Bottom (C,D): Dose-response curves for V_max (limiting activity at high Ca). Left (A,C): Cardiac SR. Right (B,D): Skeletal SR. Mean ± S.E. (n≥ 3).

Figure 5. Effects of hit compounds on cardiomyocyte contractility and Ca²⁺ transients

(A) Effects of compounds (10 μM) on the amplitude of sarcomere length oscillations (n=20, p-values indicated). (B) Ca²⁺ transients measured by fura-2, as affected by 10 μM CDN1001. (C) Mean value of transients recorded as in B (n = 20, p ≤ 0.01).

Figure 6. Screening with fluorescence lifetime plate-reader

(A) FLT decays of IAEDANS-SERCA samples in a 384-well plate. (B) Plots of intensity (gray, integrated area) and lifetime (black), with coefficient of variance (CV, indicating the well-to-well variability = σ/mean) indicated. (C) Z′ vs signal window, based on the CV values in B.