Figure 1. Dual targeting of VEGFR2 and CDK1.

(A) Chemical structure of the aminotriazole-based compound JK-31. A full description of the chemical synthesis can be found in the Materials and Methods section. (B) In silico molecular modeling of JK-31 (magenta structure) in the VEGFR2 and CDK1 kinase domains. A homology model of CDK1 was created using a structurally-related family member (CDK2 PDB code: 3s2p). The CDK1 and VEGFR2 kinase domain sequences were aligned and overlapped. JK-31 was docked into the overlapped crystal structure using Glide program and important amino acid residues identified. VEGFR2 crystal structure is shown in yellow and residues annotated in bold; CDK1 crystal structure is shown in blue/grey and residues annotated in italics. (C) JK-31 was docked into a VEGFR2 crystal structure alone (PDB code: 3cjg) and predicted hydrogen bond contacts identified. (D) JK-31 was docked into the CDK1 crystal structure homolog alone and predicted hydrogen bond contacts identified. Dotted lines denote predicted hydrogen bond contacts. Green structures denote important kinase domain residues. Further details of these models can be found in Figure S2. (E–F) JK-31 inhibits the intrinsic kinase activity of both CDK1 and VEGFR2. IC₅₀ curves were generated by incubating recombinant protein kinases with a peptide substrate, radiolabeled [γ-³³P]-ATP and either JK-31 or bohemine (5 nM to 100 µM). Effects of JK-31 and bohemine on phosphate transfer from a (E) recombinant human VEGFR2 to a peptide substrate, poly[Glu∶Tyr] (4∶1) and (F) recombinant human CDK1/cyclin B complex to histone H1 substrate in vitro. Line graphs of three independent replicate experiments are shown.