With this assay, the ADP produced by the Syk activity was converted to ATP, which is the substrate of luciferase, consequently leading to the production of light. an IC50 of 1 1.64 M (Number 4). Open in a separate window Number 3 The luminescence ideals Sclareolide (Norambreinolide) of the Syk remedy after incubation with 18 test compounds in ADP-GloTM kinase assays. The luminescence value was recognized in the presence of 1 ng/L Syk incubated with 18 compounds (30 M in the total reaction system) using an ADP-GloTM kinase assay kit for primary testing. Information about compounds 1 to 18 can be found in Table 1. Compounds 19 and 20 represent thepositive and bad control, respectively. The error bars indicate the standard error (SE) of three replicates. *** means < 0.001. Open in a separate window Number 4 The dose-response curve of tanshinone I inhibition of Syk activity. All error bars symbolize the SE of three replicates. 2.3. Tanshinone I Dose-Dependently Inhibited Mast Cell Degranulation To evaluate the anti-mast cell degranulation activity of tanshinone I, the release rate of -hexosaminidase, an important biomarker in degranulation, was measured in RBL-2H3 cells after antigen activation. Chloroquine, Sclareolide (Norambreinolide) a known mast cell degranulation inhibitor, was used like a positive control . As demonstrated in Number 5A, chloroquine (positive control) and 2.22C60.00 micromoles of tanshinone I significantly inhibited -hexosaminidase release in IgE/BSA-stimulated RBL-2H3 cells. The half-inhibitory concentration for the inhibition of Syk by tanshinone I had been determined to be 2.76 M (Figure 5B). All experiments at each concentration of tanshinone I had developed three replicates and were repeated three times. Open in a separate window Number 5 The inhibition of Syk activity by different concentrations of tanshinone I (A) and dose-response curve analysis (B). All error bars symbolize the SE of thethree replicates. ** means < 0.01 and * means < 0.05. 2.4. Binding Site of Tanshinone I in Syk Model Most of the Rabbit polyclonal to HSD17B13 known Syk inhibitor molecules have specific structural scaffolds, such as pyridine-2-carboxamide, pyrazin-8-amine, pyrimidine-8-carboxamide, pyrimidin-4-one, pyridazine-3-carboxamide, pyrimidine-5-carboxamide, (3(Danshen), a well-known traditional natural medicine in China that has a variety of pharmacological effects, including antioxidant, anti-inflammatory, heart-protective, and anti-osteoporotic effects [26,27]. Studies have found that tanshinones have anti-inflammatory, anti-allergic, and additional pharmacological effects [28,29]. Choiet al. reported that tanshinones probably exert their anti-allergic activities by influencing FcRI-mediated tyrosine phosphorylation of ERK and PLC2 . Buyanravjikh et al. reported that cryptotanshinone, a natural compound extracted from Bunge, experienced an inhibitory effect on IgE/antigen-mediated mast cell degranulation through the inhibition of tyrosine kinase-dependent degranulation signalling pathways . This study demonstrates, for the first time, that tanshinone I is definitely a direct Syk inhibitor and offers anti-mast cell degranulation activity in vitro, which may provide a perspective for elucidating the molecular mechanism of tanshinone I for its anti-allergic and additional pharmacological effects. To further evaluate the reliability of our VS workflow, a retrospective assessment was carried out . As demonstrated in the Supplementary material (Sections S1 and S2), simpler ligand-based Sclareolide (Norambreinolide) methods such as fingerprint similarity search and 3D pharmacophore model screening showed a low potency in identifying Tanshinone I from your natural compound database. Virtual testing based on Surflex-Dock not only increases the probability of identifying active compounds targeting Syk, but also predicts the connection between the bioactive molecule and target protein. 3. Materials and Methods 3.1. Molecular Docking Molecular docking was carried out using the Surflex-Dock module in the SYBYL-X 1.3 software (Tripos, Inc., St. Louis, MO, USA) [32,33,34,35]. All 320 molecules from our in-house natural compound database were downloaded from your PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in mol2 file format. All hydrogen atoms were added, and the partial atomic charges of the atoms of each compound were assigned using the Gasteiger-Hckel method. Each structure was energy-minimized using the Tripos pressure field having a distance-dependent dielectric constant and the Powell conjugate gradient algorithm convergence criteria, which partially accounts for the shielding effects of the aqueous environment on electrostatic relationships . These conformations were used as starting conformations to perform molecular docking. The crystal structure of Syk (PDB ID: 4PUZ), determined by X-ray diffraction at a 2.09 ? resolution, was chosen like a docking protein model ..