Fluorescence Polarization Assay

Fluorescence polarization (FP) assays were performed as described (Manoharan et al., 2019). The IC₅₀ of compounds were determined in a binding/displacement assay using fluorescein-labeled PMCA peptide (derived from plasma membrane Ca²⁺ transporting ATPase, a CaM binding protein) as the probe and recombinant bovine calmodulin (cat. no. 208690, Merck), which has an amino acid sequence identical to the human isoform. The F-CaMKII peptide was used at 5 nM concentration with 50 nM of His-tagged wt and mutCaM. FP assays were carried out in a black low volume round bottom 384-well plate (cat. no. 4514, Corning) with a reaction volume of 20 μl. Compounds were threefold-diluted in an assay buffer (20 mM Tris Cl pH 7.5, 50 mM NaCl, 1 mM CaCl₂, and 0.005% Tween 20), and a complex of 100 nM CaM and 10 nM F-PMCA peptide was added. The FP signals were recorded on the Clariostar (BMG labtech) plate reader with excitation at 482 ± 8 nm and emission at 530 ± 20 nm at 25°C, after 30–60-min interval for up to 5 h. Then the plate was incubated overnight at 4°C, and the next day, final readings were taken after a total of 24 h incubation. The fluorescence anisotropy was calculated and plotted against the logarithm of the compound concentration and fit to the log inhibitor vs. response–variable slope (four parameters) equation in Prism (GraphPad). The IC₅₀ of the inhibitor was converted into K_d as described in Sinijarv et al. (2017) using the equation

where [I]₅₀ is the concentration of the free inhibitor at 50% displacement, given as [I]₅₀ = IC₅₀−[EI]₅₀, where [EI]₅₀ is the concentration of the CaM:inhibitor complex in case of 50% displacement; [P]₅₀ is the concentration of the free probe at 50% displacement; [E]₀ is the concentration of free CaM at 0% displacement; and K_D,probe is the dissociation constant of the complex of the probe and CaM. The K_D of the probe, F-PMCA to CaM, is 6 nM (Manoharan et al., 2019).

The potency of the irreversible covalent inhibitors was assessed as described in Singh et al. (2011). The potency and selectivity of a covalent inhibitor are governed by two parameters, namely, K_i, the dissociation constant of the initial non-covalent complex, and k₂, the rate of the subsequent covalent bond-forming reaction as given in the chemical equation

E and I denote a protein target and its covalent inhibitor, respectively. E ⋅ I is the initial non-covalent complex, and E – I is the final covalent complex. To obtain the K_i and k₂ rates, the fluorescence polarization signal after inhibitor treatment was plotted against the incubation time and fit using a one-phase decay function to obtain the observed rate constant, k_obs. This was repeated for several inhibitor concentrations. Then, k_obs was plotted against the concentration of the inhibitor, and the data were fit to a hyperbolic equation kobs=k2×[I]Ki+[I]to obtain K_i and k₂. The ratio of k₂/K_i represents the second-order rate constant of the reaction of the covalent inhibitor with the target.