Figure 1.
Complex structure of Shh-Hhip and sequence analysis of Hhip L2 with various species of Ptchs.
(a) The crystal structure of human Shh-Hhip complex (PDB ID: 3HO5). Shh, Hhip, and Hhip L2 (M373-D387) are represented in magenta, cyan, and green colors. The solvent molecules were omitted for clarity. (b) Hydrogen bond interactions between the Shh pseudo-active site (magenta) and Hhip L2 (green) are shown in black dotted lines and the hydrogen bonding residues are displayed as stick model. (c) The result of multiple sequence alignment between Hhip L2 and various species of Ptchs [21].
Figure 2.
Comparison of the binding of loop peptides at the pseudo-active site of Shh.
(a) RMSD plots for Shh backbone during 2 ns MD simulations for following complex structures: Shh-Hhip L2 complex with all metal ions, salmon; Shh-PL2 with all metal ions, green; Shh-PL2 with no zinc ion, yellow; Shh-PL2 with no calcium ions, cyan; Shh-PL2 with no metal ions, magenta. All loop structures also painted according to the colors of RMSD plots. (b) Superimposition of final snapshots of the Shh-Hhip L2 and Shh-PL2 complexes. Electrostatic potential surface was calculated from the Shh of Shh-PL2 structure. The zinc and calcium ions are shown as bluish-gray and green spheres. (c) The PL2 forming hydrophobic residues are shown by the space-filling model. (d) Superimposition of all complex structures of the Shh-PL2 with different composition of the metal ions. Electrostatic potential surface was calculated from the Shh of Shh-PL2 structure.
Figure 3.
Pie charts of the clusters for backbone conformations of the Shh.
(a) The highly populated cluster (cluster 5) of Shh-PL2 is colored in slashdot green. (b) The highly populated cluster (cluster 2) of the Shh-robotnikinin is colored in gold.
Figure 4.
Design of structure-based pharmacophore models.
The metal ions and key residues of the representative Shh structure of (a) the Shh-PL2 (slashdot green) and (b) the Shh-robotnikinin (gold). The zinc and calcium ions are marked by blue-gray and green spheres, and the key residues are represented by stick model. Chemical features are color coded as follows: HBA, green; HBD, magenta; HYP, cyan. Selected chemical features based on the representative Shh structure generated from (c) the Shh-PL2 and (d) Shh-robotnikinin. Completed pharmacophore models of (e) the Shh-PL2 and (f) Shh-robotnikinin.
Figure 5.
Flow chart of virtual screening procedure used in the study.
Figure 6.
Final hit compounds and their pharmacophore overlay.
The hit compounds depicted in stick representation. 2D chemical structures of (a) Hit 1 (BAS 13382537) and (b) Hit 2 (BAS 06350510). Mapping of (c) Hit 1 and (d) Hit 2 upon Pharm-P. Mapping of (e) Hit 1 and (f) Hit 2 upon Pharm-R.
Table 1.
Overlapping binding modes of the hit compounds between the Shh-PL2 and Shh-robotnikinin derived pharmacophore models.
Figure 7.
Comparison of binding modes of the hit compounds.
Hit 1 and Hit 2 are depicted in yellow and green stick representations. Electrostatic potential surfaces were calculated from representative Shh structures of (a,c) the Shh-PL2 and (e,g) Shh-robotnikinin. 2D diagrams for interactions of hit compounds with the representative Shh structures of (b,d) the Shh-PL2 and (f,h) Shh-robotnikinin. Blue dashed lines indicate hydrogen bonding interactions between protein and ligand.