SBL-JP-0004: A promising dual inhibitor of JAK2 and PI3KCD against gastric cancer

Abstract

Background: Gastric cancer (GC) remains a global health burden and is often characterized by heterogeneous molecular profiles and resistance to conventional therapies. The phosphoinositide 3-kinase and PI3K and Janus kinase (JAK) signal transducer and activator of transcription (JAK-STAT) pathways play pivotal roles in GC progression, making them attractive targets for therapeutic interventions.

Methods: This study applied a computational and molecular dynamics simulation approach to identify and characterize SBL-JP-0004 as a potential dual inhibitor of JAK2 and PI3KCD kinases. KATOIII and SNU-5 GC cells were used for in vitro evaluation.

Results: SBL-JP-0004 exhibited a robust binding affinity for JAK2 and PI3KCD kinases, as evidenced by molecular docking scores and molecular dynamics simulations. Binding interactions and Gibbs binding free energy estimates confirmed stable and favorable interactions with target proteins. SBL-JP-0004 displayed an half-maximal inhibitory concentration (IC₅₀) value of 118.9 nM against JAK2 kinase and 200.9 nM against PI3KCD enzymes. SBL-JP-0004 exhibited potent inhibition of cell proliferation in KATOIII and SNU-5 cells, with half-maximal growth inhibitory concentration (GI₅₀) values of 250.8 and 516.3 nM, respectively. A significant elevation in the early phase apoptosis (28.53% in KATOIII cells and 26.85% in SNU-5 cells) and late phase apoptosis (17.37% in KATOIII cells and 10.05% in SNU-5 cells) were observed with SBL-JP-0004 treatment compared to 2.1% and 2.83% in their respective controls.

Conclusion: The results highlight SBL-JP-0004 as a promising dual inhibitor targeting JAK2 and PI3KCD kinases for treating GC and warrant further preclinical and clinical investigations to validate its utility in clinical settings.

Keywords: Dual inhibition; Gastric cancer (GC); JAK2; Molecular docking; Molecular dynamics simulation; PI3KCD; SBL-JP-0004.

Figure 1. Structure of JAK2 and PI3KCD. It reveals druggable pocket. (a) Theoretical structure of JAK2 kinase, colored based on secondary structure. (b) Presence of ATP binding pocket (active site) kinase domain of JAK2, green contour representation, showing amino acid residues surrounding the cavity. (c) Crystal structure of PI3KCD kinase, colored based on secondary structure, bound to a reference ligand (85S). (d) protein-ligand interaction analysis showing key amino acid residues of PI3KCD involved in the interactions with 85S.

Figure 2. High-throughput Virtual Screening of ChemBridge library. (a) Predicted docking scores based on high-throughput Virtual Screening of ChemBridge library against JAK2 kinase domain, showing top-ranked ligands. (b) predicted docking scores for molecules binding to both JAK2 and PI3KCD kinase. (c) 2D structure of SBL-JP-004.

Figure 3. Protein-ligand interaction analyses. (a) Predicted binding pose for SBL-JP-0004 with JAK2. (b) Protein-ligand interaction analysis profiling shows amino acid residues of JAK2 in contact with the lead molecule SBL-JP-0004. (c) Predicted binding pose for SBL-JP-0004 with PI3KCD kinase. (d) Protein-ligand interaction analysis profiling shows amino acid residues of PI3KCD kinase in contact with the lead molecule SBL-JP-0004.

Figure 4. Molecular dynamic simulation predicts protein-ligand stability. (a) Snapshot of simulation trajectories captured before and after 100 ns simulation, depicting SBL-JP-0004 binding to JAK2. (b) Ligand Root Mean Square Deviation (RMSD) of SBL-JP-0004 in complex with JAK2, depicting binding stability. (c) Snapshot of simulation trajectories captured before and after 100 ns simulation, depicting SBL-JP-0004 binding to PI3KCD kinase. (d) Ligand Root Mean Square Deviation (RMSD) of SBL-JP-0004 in complex with PI3KCD kinase, depicting the binding stability.

Figure 5. Binding free-energy estimation. MM-PBSA-based binding free energy estimate shows the average Gibbs binding energy estimate between SBL-JP-0004 and (a) JAK2 and (b) PI3KCD kinase, calculated from 100 ns simulation trajectories.

Figure 6. Biological efficacy of SBL-J2P-004. The enzyme IC₅₀ values of SBL-JP-0004 against JAK2 (a) and PI3KCD (b) are shown. These values represent the mean ± standard deviation (SD) from three independent experiments, with IC₅₀ values determined using GraphPad Prism version 6.0 software. Additionally, the GI₅₀ values for cell proliferation in KATOIII and SNU-5 cells treated with SBL-JP-0004 (c), as well as the impact of the compound on the viability of Vero cells at various concentrations (d), are illustrated. Cell proliferation and viability were assessed using the MTT assay, with mean ± SD values analyzed using GraphPad Prism version 6.0 software, *p < 0.05.

Figure 7. Early and late phase apoptosis induction by SBL-JP-004. The compound induced apoptosis: (a) Annexin V assay results showing the proportions of early and late apoptotic cells in KATOIII and SNU-5 cells following treatment with SBL-JP-0004 are presented. (b) The compound increased both early and late apoptotic populations in these cells after 48 h of treatment. Each experiment was performed in triplicate, and the results shown are representative. Data are expressed as mean ± SD. *p < 0.05.