Figure 1.
Components of Src and its activation mechanisms.
(A) SH2 binds to the phosphorylated Tyr530 and SH3 binds with prolines on the linker domain, effectively locking the Src in an inactive closed conformation. (B) Src is activated when Tyr530 is dephosphorylated and Tyr416 within the catalytic domain is autophosphorylated.
Figure 2.
Scaffolds of Bosutinib, Dasatinib, amd Saracatinib, and their respective status in clinical trials.
Figure 3.
Structural scaffolds of TCM candidates and Saracatinib.
(A) Saracatinib, (B) Isopraeroside IV, (C) 9HFG, and (D) aurantiamide.
Table 1.
DockScore and related attributes of Sacracatinib and top three TCM candidates calculated by Discovery Studio 2.5 (D.S. 2.5).
Figure 4.
ADMET adsorption model of Saracatinib and the TCM candidates generated by DS 2.5.
Table 2.
Adsorption, distribution, metabolism, toxicity properties and predicted pIC50 of Saracatinib and the top three TCM compounds.
Figure 5.
Docking poses of different ligands in Src kinase ATP binding pocket.
Shown are snapshots of (A) Saracatinib,(B) Isopraeroside IV, (C) 9HFG, and (D) aurantiamide during docking simulation with Src kinase. Purple and blue surfaces are added to denote the small lobe (267–337) and large lobe (340–520) of Src kinase, respectively. Hydrogen bonds are shown as dotted green lines and pi-interactions are shown in orange. (A) Pi-interactions with Lys273 and Lys295 are critical for Saracatinib. (B) Isopraeroside IV docks to the outer region of the ATP binding pocket via H-bonds at Ser345 and Asp348. (C) 9HFG structure enables docking in the inner regions of the ATP binding pocket, forming H-bonds with Lys295 and Asp404. (D) Similar to Saracatinib, aurantiamide forms pi-interactions with Lys295, in addition to H-bonds with Ser345 and Asp348.
Figure 6.
Ligplot diagrams illustrating protein-ligand interactions during docking.
(A) Saracatinib, (B) Isopraeroside IV, (C) 9HFG, and (D) aurantiamide. Hydrophobic interactions are represented by red spokes radiating towards the ligand atoms they contact. Ligands are represented in purple. C, N, O, and Cl atoms are represented in black, blue, red, and green, respectively. Pink backgrounds highlight amino acids of the small lobe (267–337), and blue background highlight those belonging to the large lobe (340–520). Hydrophobic interactions shown in this illustration are calculated through the Ligplot algorithm.
Table 3.
Ligand-protein interactions determined through LigPlot.
Figure 7.
Correlation between observed and predicted activities of 53 Src inhibitors using different prediction models.
(A) MLR and (B) SVM. Correlation coefficients (R2) calculated from the training set were 0.837 and 0.7922 for MLR and SVM models respectively.
Table 4.
CoMFA and CoMSIA models constructed from 53 known Src kinase inhibitors1.
Figure 8.
Correlation between observed and predicted activities of 53 Src inhibitors using different 3D-QSAR models.
(A) CoMFA and (B) CoMSIA. Correlation coefficients (R2) calculated from the training set were 0.8448 and 0.9014 for CoMFA and CoMSIA models respectively.
Figure 9.
Spatial contour of test ligands to generated CoMFA maps.
Map areas favoring and disfavoring steric fields are represented respectively in green and yellow. Saracatinib (A), Isopraeroside IV (B), and 9HGF (C) contoured well to the bioactivity map. A benzene ring of aurantiamide was located within the steric disfavoring region (D), suggesting lower bioactivity than it other tested ligands.
Figure 10.
Spatial contour of test ligands to generated CoMSIA maps.
Color blocks represent favor/disfavor steric field (green/yellow), favor/disfavor hydrophobic interaction (cyan/white), favor/disfavor hydrogen donor (purple/magenta). Saracatinib (A) forms H-bonds with Met341 which is located near the region favoring hydrogen bond donors and hydrophobic interactions. The compact structures of Isopraeroside IV (B) and 9HGF (C) allowed interactions with Src kinase without defying regions disfavoring interactions. The benzene moieties of aurantiamide (D) were in proximity to the steric disfavoring region, suggesting lower bioactivity.
Figure 11.
Hydrogen bond distance profiles between Src kinase and TCM candidates during 20 ns MD simulation.
Distances (Å) shown are profiles of amino acids implicated as binding residues during docking with (A) Saracatinib, (B,C) Isopraeroside IV, (D–G) 9HFG, and (H) aurantiamide.
Figure 12.
Molecular changes contributing to the disappearance of pi interactions in Saracatinib and aurantiamide during MD.
Loss of pi-interaction with Leu273 in both ligands was due to torsion of the benzene ring within the ligand structures.
Table 5.
Summary of interaction type, location, and frequency of test ligands following docking and MD simulation.
Table 6.
H-bond distance and occupancies1 of different ligands during MD.
Figure 13.
Molecular changes contributing to protein-ligand complex stability during MD.
(A) Lys343 and Ser345 amine group rotations bring Isopraeroside IV into proximity for H-bond formation. (B) The rhythmic fluctuations observed in 9HFG can be attributed to the rotations of the NH3 group on Lys295. (C) Rotation of the benzene moieties led to increased distance fluctuations in Ser345 during MD.
Figure 14.
Torsion angles of test candidates during MD simulation.
Torsion angle measured is designated by a lower case alphabet which corresponds to the radar chart with the same alphabet. Red numbers indicate the locations where H-bonds are formed. (numbering corresponds to those in Table 5). Blue lines indicate the torsion angles recorded; the red and gray lines indicate the angle during docking and at time 0 of MD, respectively.
Figure 15.
Measured trajectories during MD.
(A) Complex, (B) ligand, and (C) total energy during MD. Saracatinib had the highest complex RMSD and total energy. The lower complex RMSDs and total energy of Isopraeroside IV and 9HFG may be attributed to their compact structure and multiple H-bond formations. The higher RMSD and total energy observed in auranruamide is possibly the result of the rotating benzene moieties.
Figure 16.
Mechanisms underlying Src kinase inhibition and activation.
(A) Based on the molecular interactions observed through MD, TCM candidates 9HFG and aurantiamide (A; violet diamond) may inhibit Src kinase activation through different pathways. Aurantiamide binds directly to Lys295, competing with ATP for the binding site. 9HGF also binds to Lys295, but has additional binding with Asp404, which acts similarly to Mg2+ found in native Src kinase. (B) The bridging function of Mg2+ between Lys295 and Asp404 in native Src kinase.