Discovery and biological evaluation of a novel and highly potent JAK2 inhibitor for the treatment of triple negative breast cancer

Abstract

Janus kinase 2 (JAK2) is considered an attractive target for the treatment of triple-negative breast cancer (TNBC). Herein, we discovered six JAK2 inhibitors using structure-based virtual screening and molecular docking. Among them, JNN-5 was the best compound. It indicated strong inhibitory effects on JAK2 in the nanomolar range (IC₅₀ = 0.41 ± 0.03 nM), and high selectivity for JAK2 over JAK1 and JAK3 (selectivity index (SI) > 73.17). Moreover, molecular dynamics (MD) simulation exhibited that JNN-5 bound with high stability to JAK2 JH1. Cellular assays revealed that JNN-5 displayed strong antiproliferative activities in the TNBC cell lines (MDA-MB-468, MDA-MB-213, HCC70, MDA-MB-157). JNN-5 significantly reduced the migration of HUVECs with the dose-dependence. JNN-5 had a significant inhibitory effect on multidrug-resistant MDA-MB-231/ADR (IC₅₀ = 0.37 ± 0.02 μM). These data demonstrate that JNN-5 may be a highly effective and selective antitumor compound for the treatment of TNBC.

Keywords: JAK2; Triple negative breast cancer (TNBC); biological evaluation; inhibitor; structure-based virtual screening.

Figure 1.

Pharmacophore model based on JAK2 JH1. The pharmacophore model generated using the MOE tool exhibited pharmacophore features as follows: F1 and F5 were aromatic centres, displayed in yellow; F2 was an aromatic centre, displayed in orange; F3 and F6 were hydrogen bond acceptors, displayed in cyan; F4 was a hydrogen bond donor, displayed in purple. The protein was coloured in gray, and the key residues within the active site of the JAK2 JH1 were displayed in stick representation with three-letter amino acid codes. Carbon atoms were coloured gray, oxygen atoms were red, and nitrogen atoms were blue. Hydrogen bonds were represented by black dashed lines.

Figure 2.

A flowchart of the identification of JAK2 inhibitors via an integrated strategy of in silico screening and biological evaluation.

Figure 3.

The docking scores of JNNs 1–6 and CYT-387, n = 3, ***p < 0.001 vs CYT-387.

Figure 4.

The chemical structures of JNNs 1–6.

Figure 5.

The predicted docking poses and binding surface of JNNs 1–3 in the active site of JAK2 JH1, respectively. (A and B) represented JNN-1, with ligand colours being green; (C and D) represented JNN-2, with ligand colour being purple; (E and F) represented JNN-3, with ligand colour being orange. The binding surface of JAK2 JH1 were represented by hydrogen bond regions (green), hydrophobic regions (white), and moderately polar regions (red), presenting the narrow and elongated area. The predicted docking poses and binding surface of JNNs 4–6 in the active site of JAK2 JH1, respectively. (G and H) represented JNN-4, with ligand colour being pink; L and M represented JNN-5, with ligand colour being blue; N and Q represented JNN-6, with ligand colour being brown.

Figure 5.

The predicted docking poses and binding surface of JNNs 1–3 in the active site of JAK2 JH1, respectively. (A and B) represented JNN-1, with ligand colours being green; (C and D) represented JNN-2, with ligand colour being purple; (E and F) represented JNN-3, with ligand colour being orange. The binding surface of JAK2 JH1 were represented by hydrogen bond regions (green), hydrophobic regions (white), and moderately polar regions (red), presenting the narrow and elongated area. The predicted docking poses and binding surface of JNNs 4–6 in the active site of JAK2 JH1, respectively. (G and H) represented JNN-4, with ligand colour being pink; L and M represented JNN-5, with ligand colour being blue; N and Q represented JNN-6, with ligand colour being brown.

Figure 6.

Cell inhibition rates of JNNs 1–6 and CYT-387 at 1 μM, n = 3, ***p < 0.001 vs CYT-387.

Figure 7.

MD simulation of JAK2 JH1 in complex with JNN-1 and JNN-5. (A and B) The backbone RMSD of the complex of JAK2 JH1 and JNN-1/JNN-5, respectively. (C and D) RMSF of JAK2 JH1 Cα atoms in the complex of JAK2 JH1 and JNN-1/JNN-5, respectively. (E and F) The secondary structures analysis of JAK2 JH1 in the complex of JAK2 JH1 and JNN-1/JNN-5, respectively. (G and H) Rg of JAK2 JH1 in the complex of JAK2 JH1 and JNN-1/JNN-5, respectively.