Within the crystal structures of Clk115,47 and Dyrk1A,16,48 a hydrogen bond between a ligand and Lys191 in Clk1, a residue from a B sheet on 1 side of the ATP binding cleft is very important for ligandprotein interaction. Similarly, binding model obtained by docking ligands for the ATP binding domain of Clk4 indicated that the corresponding residue Lys 189 in Clk4 formed hydrogen bonds with all three ligands. Compound 1 has the highest inhibition activity amongst all tested compounds. Above described 3D QSAR model indicated that a hydrophobic R1 substitute around the position 4 amine is favorable. Figure 5B represented that the methyl group on compound 1 is oriented into a hydrophobic pocket surrounded by the side chains of residues Val173, Ala187, and Phe239, which could increase the van der Walls interaction among compound 1 and Clk4. Compound 29 was selected as a chemical probe for Clk4 that has selectivity of Clk4 against other Clk and Dyrk.
12,13 Figure 5C showed that there is a hydrogen bond among the hydroxyl group around the R3 substituent of compound 29 plus the side chain of Asp248, which could contribute for the selective inhibitory eects of this compound against Clk4. The superimposing in between structures of Dyrk1A and Clk4 is shown on Figure 5D. In comparison to the side chain of Asp248 of Clk4, the corresponding atoms of residue Asp247 in purchase MK-0752 Dyrk1A are moving away in the binding pocket by about 2, which could account for the higher selectivity of this compound in between Clk4 and Dyrk1A. The interaction among Clk4 and ligands identied by docking agreed with the outcomes from ligand primarily based pharmacophore and 3D QSAR models. The hydrogen bond involving side chain of Lys189 along with the nitrogen of quinazoline ring of compounds 1, 29, and 52 was consistent with the hydrogen bond donor feature located on the position 1 nitrogen of quinazoline core identied by the pharmacophore model featured within this study.
The orientation with the hydrophilic selleck R3 substituent of compound 29 and 1 for the hydrophilic pocket of Clk4 was supported by the contour maps obtained through 3D QSAR model indicating that hydrophilic and electron withdrawing groups were favored within this area. By contrast, the unfavorable interaction between the hydrophobic methylphenyl group of compound 52 and the hydrophilic pocket could account for its much reduced inhibitory activity than compound 1 and 29. It is noticed that there had been two hydrogen bond donors featured on the nitrogen atoms of quinazoline ring by the pharmacophore model but only 1 of them participated in the hydrogen bonding interaction with Clk4. Pharmacophore traits are indications of structural properties of ligands interacting with a receptor but do not necessarily determine crucial attributes which are accountable for ligandprotein interaction.