(一)
A non-radioactive, sensitive, rapid and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells only required about 2.5 mg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of Km and Vmax were 6.1 ± 0.3 mM and 135.4 ± 1.5 nmol AdoMet formed/mg protein/h, respectively.
(二)
Molecular docking is an increasingly important method for computer-aided drug design, and scoring functions play an essential role in docking strategy. However, high false-positive rates remain a common issue in structure-based virtual screening. In this study, we developed a pharmacophore-based, knowledge-based and empirical-based scoring function (HotLig) to predict protein-ligand interactions. Connolly surface of binding pocket was constructed by PscanMS first and was used to estimate the hydrophobic effect between protein and ligand. The distance-dependant potential for polar interactions (including H-bond, ionic bond and metal bond) and hydrophobic effect were derived from 600 complexes in protein data bank (PDB) according to the statistic frequency of each molecular interaction. Then the energy parameter of each interaction was determined by 214 complexes of training set. The training set was classified into three subsets: basic set (101 complexes), ionic set (56 complexes) and metal set (57 complexes). The Wang 100 dataset and GOLD 100 dataset were used for evaluation of prediction of binding modes. The success rate (RMSD £ 2 Å) for Wang 100 dataset and GOLD 100 dataset were 91% and 87%, respectively. The Wang 100 dataset was also used for evaluation of prediction of binding affinities. The Spearman correlation coefficient (Rs) was 0.6091. In the evaluation of virtual screening, thymidine kinase (PDB code: 1kim) was used for docking. The ligand database contained 1000 decoys and 10 actives. All submicromolar actives were found in the top 25 ligands of ranked database. Thus, HotLig predicts the interactions between protein and ligand with high accuracy, and provides a novel choice of scoring function in molecular docking studies.
