2.7 FAS Assay
FAS assay was performed according to Li et al . (2002) with modifications. Crude protein was made up to a concentration of 0.5 mg/mL for the assay. Assay was carried out in 96 well plates with the final working volume of 100 μL. Compounds to be tested were incubated with previously made crude protein extract for 30 min at 25 °C. 50 μL of this mixture was added to 50 μL of reaction mixture containing 1 mM each of malonyl CoA and NADPH, 40 μM acetyl CoA, and 2 mM DTT in phosphate buffer (250 mM). The microplate was read immediately for 10 min at 340 nm. FAS activity was calculated by subtracting the optical density (OD) obtained at 1 min from the OD obtained at 10 min. FAS activity was calculated and compared with that of control and test compounds. DMSO was taken as control as it was used to dilute the compounds. The IC50 was determined by linear regression. The negative control consisted of protein plus reaction buffer. DMSO (1 %) was also tested and it did not interfere with FAS activity. Another control consisted of buffer reaction without protein.
FAS activity was determined by the rate of oxidation of NADPH to NADP which was monitored at 340 nm. The change in concentration of NADPH during oxidation was calculated using the following equation:
ΔC=ΔA/E
Where, ΔC was change in the concentration of NADPH, ΔA was change in absorbance, and E was extinction coefficient of NADPH (E340nm = 6.22 mM−1∙cm−1). FAS activity was expressed as nM NADPH oxidized/min/mg protein.
2.8 Molecular docking studies
Molecular docking studies were carried out to explain the possible binding mode of the purified compound to FAS. Since the structure of the purified inhibitor (Phormidin) was similar to Cerulenin, the crystal structure with protein data bank (PDB) ID 4LS7 was selected as receptor which is of Bacillus subtilis FAS II beta-ketoacyl-ACP synthase II (FabF) domain, with non-covalently bonded Cerulenin. Similarly, for studying the interactions of the ligand with mammalian FAS I, PDB 3HHD (Pappenberger et al., 2010) consisting of human FAS-I’s KS-MAT domain was taken as model. Prior to the docking studies, the receptor structure was prepared by deleting the crystallographic water molecules and adding hydrogen to polar moities. Later, the disulphide bond lengths were corrected and a minimization was performed by applying a RMSD cut off 0.30 Å using a force field OPLS 2001. In the PDB structure of 4LS7 (Trajtenberg et al. , 2014), Cerulenin at the active site was taken as reference and grid was generated with 15 x 15 x 15 Å volume surrounding the ligand. In the case of PDB structure 3HHD, according to Von Wettstein-Knowles et al. (2006), active site residues Cys161, His331 and His293 play crucial roles in FAS I’s activity. Hence, these residues were taken as the centroid and grid was generated with a box of 15 x 15 x 15 Å. Docking simulation was performed using extra precision (XP) docking method implemented in Schrodinger version 9.4.0 (Friesneret al. , 2006).
2.8.1 Calculation of binding free energies
The docked poses with highest glide score were selected and binding free energies for the protein-ligand complex were calculated using Prime MM-GBSA (Friesner et al. , 2006). Prime MM-GBSA generates energy properties for the ligand, the receptor, and complex structure along with energy differences relating to strain and binding. Prime calculates the binding free energy using the equation:
ΔG (bind) = E_complex (minimized) - (E_ligand (minimized) + E_receptor (minimized))