HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin

Figures

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  Fig. 1.. HTS Assay design. (A) Schematic depicting key structural and functional states (Working, Relaxed, Super-Relaxed) of cardiac muscle myosin based on current models of cardiac myosin thick-filament structure and function [18, 40]. (B) Adaptation of a FRET sensor described in detail in Rhode [9] et al. to cardiac myosin synthetic filaments used in HTS. (C) HTS workflow (ordered top to bottom) using the FRET assay to screen chemical libraries (HTS, high-throughput screening).

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  Fig. 2.. HTS pilot screen. (A) Assay response to control compounds OM and (B) Mavacamten. (C) 60 K pilot screen lifetime. (D) Plate set (colors indicate successive assay runs) robust Z-score mean = 0.87, std = 0.02, min = 0.78, max = 0.90). (E) Pilot screen Hit retest. Confirmed effector compounds localize to upper right and lower left quadrants. Diagonal solid line in panel E indicates linear fit to primary screen Hit delta moment data (Y axis, picoseconds) and Hit retest delta moment data (X axis, picoseconds). Dashed line in panel E indicates slope = 1.0 for reference. (F) Histogram of pilot screen lifetimes. Dashed lines indicate Hit selection thresholds. Solid line indicate Gaussian fit to the histogram. (G) Histogram of the pilot screen retest.

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  Fig. 3.. Impact of pilot screen confirmed retest compounds on basal myosin filament ATPase and calcium regulated myofibril ATPase activity. (A, B, C) Compounds separated into three classes based on FRET (black lines and symbols) and myosin filament ATPase activity (red symbols and lines): (A) Increase FRET like Mava and inhibit ATPase activity (B) Increase FRET like Mava and increase ATPase activity (C) Decrease FRET and increase ATPase activity. Myofibril ATPase activity measured at varied concentrations of calcium (D). X-axis pCa is the –log of the Molar free [Ca + 2]. Myofibril ATPase inhibited by Mava (E) and activated by OM (F). Representative pilot screen myofibril activators (G) or inhibitors (H) tested at free [Ca + 2] corresponding to 10 −4 Molar (pCa 4.0), 10 −6 Molar (pCa 6.0), and 10 −8 Molar (pCa 8.0).

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  Fig. 4.. Automated HTS. (A) Assay moment robust Z-score for 1.6 M compound library screen. Colors indicate independent assay runs performed on different days. (B) Mean response of the FLT assay to OM and Mava +/− standard deviation of control compounds during the 1.6 M compound library screen. (C) Fluorescence interference filter CH1/CH2 intensity ratio (y-axis) vs. FLT% Mava Control Median. Compounds that exceeded 10% increase and decrease (blue) in the CH1/CH2 ration (horizontal lines) were deprioritized. Compounds within this range that exhibited FLT > 112.5% or < 77.5% Mava control median where advanced for retest. (D) Hit retest primary screen FLT relative control median compared to retest FLT relative Control Median. Confirmed effector compounds localize to upper right and lower left quadrants. Diagonal solid line in panel D indicates linear fit to primary screen Hit change in moment relative to the median control (Mava) change in moment (Y axis) and Hit retest change in moment relative to the median control (Mava) change in moment (X axis). Dashed line in panel D indicates slope = 1.0 for reference. (E) Histogram of primary screen lifetimes. Dashed lines indicate Hit selection…

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  Fig. 5.. Myosin Filament FLT and ATPase CRC of retest positive compounds. (A) Representative FLT decreasing (top, similar to Mava) and increasing (bottom) compounds. (B) Representative filament ATPase activators (top) and inhibitors (bottom). (C) Summary of EC50 values from CRC analysis.

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  Fig. 6.. Cardiac Myofibril ATPase measured at pCa 4.0, 6.0, and 8.0 and Aorta Smooth Muscle ATPase (SMM) activity of select allosteric modulators.