Preparation of FMSP
According to our previously reported protocol, acetyl ferrocene reacted with 4-(methylsulfonyl)benzaldehyde in sodium hydroxide and ethanol to yield 1-ferrocenyl-3-(4-methylsulfonylphenyl)propen-1-one (FMSP) [19]. The synthesized compound was well characterized by elemental analysis, multinuclear (1H) NMR, and spectroscopic methods such as IR and Mass.
Molecular modeling and docking studies
Docking studies were performed using AutoDock software version 4.0 to search for favorable binding configurations between the small flexible ligands and the rigid protein to predict their interactions. These studies were performed based on the high-resolution crystal structure of calmodulin receptor (PDB code: 1QIW) retrieved from RCSB Protein Data Bank. The co-crystallized ligand (DPD) and water molecules were removed from the protein, Kollman charges were added, nonpolar hydrogens were merged, and AutoDock 4 atom type was assigned to achieve the PDBQT format of the protein. The ligand structures (Naringenin and FMSP) were minimized using HyperChem8.0 (MM+ method) and then converted to PDBQT file format with AutoDock tools. A docking grid box was built with (30×30×30) surround the co-crystallized ligand. The Lamarckian genetic search algorithm was employed, and the docking run was set to 100. Protein residues with atoms greater than 6.0 Å from the docking box were removed for efficiency. The quality of the docked structures was evaluated by measuring the intermolecular energy of the ligand–enzyme assembly.
Determination of kinetic parameters
We used ΔG (H2O) as an appropriate parameter to calculate macromolecular stability of CaM protein in the presence of FMSP and naringenin. The free energy difference (ΔG) between the native and denatured forms of CaM was calculated by below formula [20]:
ΔG =-RT Ln [Fd /(1-Fd)]
Fd = (Yn − Yobs) / (Yn−Yd) (1)
Ln [(Yn − Yobs) / ( Yobs – Yd)] = -RTLnK
In this formula, R stands for gas constant, T represents the absolute temperature, Yobs indicates fluorescence emission intensity of CaM at different concentrations of FMSP and naringenin, and Yn and Yd stand for values of native and denatured states, respectively. The changes in ΔG values were plotted against FMSP and naringenin concentrations, which showed a linear relation based on the following equation [20]:
ΔG = ΔG (H2O) – m [D] (2)
In the above mentioned formula, ΔG (H2O) stands for ΔG at zero concentration of ligand, m is the constant for the dependence of ΔG on ligand concentration, and [D] is the ligand concentration.
Lineweaver-burk plots were created by kinetic analysis of CaM-dependent PDE inhibition in the presence of FMSP and naringenin. Thereafter, in the presence and absence of these two inhibitors, Km values calculated from these plots. According to the plots, maximum velocity (Vmax) of the activated PDE1 in the presence and absence of inhibitors is the point at which the lines crossed the y-axis.
Cell culture
The K562 human erythroleukemic cell line was provided by Pasteur Institute, Tehran, Iran. Subsequently, the cancer cells were cultured in RPMI 1640 culture medium (Thermo Fisher Scientific, USA), which were supplemented with 10%FBS (Gibco, Germany), 100 unit/ml penicillin/100 mg/ ml streptomycin (Thermo Fisher Scientific, USA), and 1% L-glutamine (Gibco, Germany). The cultured cells were incubated in 5% CO2 at 37°C conditions.
Cell viability analysis
Cell viability estimation was carried out using 3-[4, 5- methylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. In brief, the cultured cells were trypsinized and collected to be seeded in 96-well tissue culture plates (5000 cells/well) overnight. Then, the cells were treated with various concentrations of FMSP (0–5 µM) and naringenin (50µM) for 48h. Subsequently, a solution containing 10% MTT was prepared using non-complete culture medium and was added to the well for 4 h. After 4-h incubation, MTT-contained medium was replaced by 100 µL Dimethyl sulfoxide (DMSO) (Sigma-Aldrich Company, USA) to solve the produced MTT crystals. Finally, the absorbance of the solution was read by using an ELISA reader at 570 nm and the cell viabilities were determined as the percentage of untreated control cells.
Quantitative determination of cAMP concentration
The concentration of cAMP molecules within the cells was measured by an enzyme immunoassay kit (Stratagene, La Jolla, CA). Briefly, after treating the K562 cells with the mentioned concentrations of FMSP and naringenin in treatment group and culture medium in controls, about 2×105 cells were collected and centrifuged at 1200 rpm and 4ºC for 5 min. Cell pellets resuspended by the lysis buffer provided in the kit. According to the kit protocols, cAMP concentration was instantly measured in lysed cells. This method was principally based on the competition between unlabeled cAMP and stable quantity of peroxidase-labeled cAMP for the limited binding sites on an antibody against cAMP molecule.
Measurement of PKA activity
In this present study, a colorimetric assay kit (BioVision, USA) was used for determination of PKA enzyme activity. Briefly, the treated K562 cells were washed with Phosphate-buffered saline (PBS) buffer (pH = 7.4) twice, and the resuspended by lysis buffer (50mMTris–HCl, 2.5mMEDTA, 1mMMgCl2, 10mMNaF, 10%glycerol, pH 7.2) and sonicated followed by centrifugation at 1200 rpm and 4ºC for 5 min. Finally, the activity of PKA enzyme was measured according to the instructions that were provided by the manufacturer.
Effects of PKA blocking on cell growth inhibition
Rp-cAMP (Adenosine 3,5-cyclic monophosphorothioate triethyammonium salt hydrate) was used as a specific inhibitor of PKA (Biolog Life Science Institute, Bremen, Germany). This molecular inhibitor was then diluted by using RPMI culture medium to prepare 100 µM Rp-cAMP. Briefly, the cultured cells were first incubated with Rp-cAMP (100 µM) for 20 min and then treated with FMSP (0–5µM) and its combination with naringenin (50µM).
Statistical analysis
To assess the normality of data and homogeneity of variances, by K–S and Levene's statistical tests were used, respectively. Then, one-way analysis of variance (ANOVA) or Mann–Whitney nonparametric tests were applied to analyze the results of the study. The statistical significance of the obtained results was considered at p < 0.05 level between the treated and control groups. Data were analyzed by using SPSS software version 22, and results were indicated as the mean ± standard error (mean ± SE).