FIGURE 1: Principle of the high-throughput fluorescence anisotropy assay. (A) Flowchart of the assay procedure for the high-throughput screening. The initial PYD solution shown in light blue is prepared by mixing the specified volume amounts from 25 µM and 1 µM stock solutions of not labelled PYD and Fluorophore-labelled PYD (PYD*) respectively and 10% Triton solution, yellow rectangles. In the premixed solution labelled and unlabelled PYD are maintained in a soluble and monomeric forms by low pH condition. The addition to premixed PYD solutions of either NaCl as positive control or DMSO solution with and without the compounds are identified by the green rectangles. The resulting assay mixture is highlighted in light blue. The jump from acidic to physiological pH condition is achieved by addition of a fixed volume amount of a 60 mM NaOH solution to the assay mixture, highlighted by the dark blue rectangle. Fluorescence anisotropy is monitored as a single point measurement after incubation for 2 h at 25^°C and filament formation is evaluated as the difference between fluorescence anisotropy at the initial and endpoint of the measurement, grey rectangles. (B) Scheme of the ASC filament formation during the fluorescence anisotropy (FA) assay. Monomeric PYD domain of ASC protein (green) and PYD* labelled with Alexa Dylight 488 fluorophore (red). The pH shift-induced filament formation leads to the anisotropy increases. (C) A schematic 2D diagram of the PYD filament arrangement where individual monomeric PYD domains required for filament formation are coloured in dark green, teal and light blue over the filament scaffold reported in light green. The asymmetric interfaces type I, II and III as observed on the human (PDB 3J63) and mouse (PDB 2N1F) PYD filament structures are labelled in white. The black arrow highlights the directionality of filament propagation within a single layer through the interactions between the interfaces type Ia and Ib.