Combining structure-based pharmacophore modeling, virtual screening, and in silico ADMET analysis to discover novel tetrahydro-quinoline based pyruvate kinase isozyme M2 activators with antitumor activity

Abstract

Compared with normal differentiated cells, cancer cells upregulate the expression of pyruvate kinase isozyme M2 (PKM2) to support glycolytic intermediates for anabolic processes, including the synthesis of nucleic acids, amino acids, and lipids. In this study, a combination of the structure-based pharmacophore modeling and a hybrid protocol of virtual screening methods comprised of pharmacophore model-based virtual screening, docking-based virtual screening, and in silico ADMET (absorption, distribution, metabolism, excretion and toxicity) analysis were used to retrieve novel PKM2 activators from commercially available chemical databases. Tetrahydroquinoline derivatives were identified as potential scaffolds of PKM2 activators. Thus, the hybrid virtual screening approach was applied to screen the focused tetrahydroquinoline derivatives embedded in the ZINC database. Six hit compounds were selected from the final hits and experimental studies were then performed. Compound 8 displayed a potent inhibitory effect on human lung cancer cells. Following treatment with Compound 8, cell viability, apoptosis, and reactive oxygen species (ROS) production were examined in A549 cells. Finally, we evaluated the effects of Compound 8 on mice xenograft tumor models in vivo. These results may provide important information for further research on novel PKM2 activators as antitumor agents.

Keywords: molecular docking; pharmacophore; pyruvate kinase; virtual screening.

Figure 1

Representative chemical structures of previously reported hPKM2 activators. Abbreviations: PKM, pyruvate kinase isozyme; AC₅₀, activation concentration 50%; hPKM2, human PKM2.

Figure 2

Virtual screening protocol for the identification of novel PKM2 activators. Abbreviations: PKM, pyruvate kinase isozyme; ADMET, absorption, distribution, metabolism, excretion and toxicity; PBVS, pharmacophore based virtual screening; DBVS, docking based virtual screening.

Figure 3

Comprehensive pharmacophore features (A) and the common structure-based pharmacophore model (B) generated by seven PKM2-activator complexes. Note: The green boxes indicate the key pharmacophore features. Abbreviations: PKM, pyruvate kinase isozyme; Ar, aromatic rings; HP, hydrophobic features.

Figure 4

Plot of PSA versus AlogP for candidate compounds showing the 95% and 99% confidence limit ellipses corresponding to the blood–brain barrier and intestinal absorption models. Note: The numbers correspond to the compounds in Table 2. Abbreviations: ADMET, absorption, distribution, metabolism, excretion and toxicity; AlogP, the logarithm of the partition coefficient between n-octanol and water; BBB, blood–brain barrier; PSA, polar surface area; 2D, two-dimensional.

Figure 5

The effects of Compound 8 on A549 lung cancer cell viability. Notes: (A) MTT assays showed Compound 8 inhibited A549 cell proliferation in a hypoxic environment in a concentration- and time-dependent manner. Data are expressed as means ± standard deviation from three independent experiments. (B) The colony clusters were detected by crystal violet staining, after a 10-day Compound 8 treatment in a hypoxic environment. (C) Statistical results of colony-forming assays presented as surviving colonies. Data are expressed as means ± standard deviation from three independent experiments (*P<0.05; **P<0.01).

Figure 6

The fluorescent thymidine analog EdU was used to identify S-phase cells by labeling their replicating DNA (red), nuclei are colabeled with DAPI (blue). Notes: (A–C) Blank vehicle under normoxia (21% O₂); (D–F) blank vehicle under hypoxia (1% O₂); (G–I) compound 8 treatment under hypoxia (1% O₂). Abbreviations: EdU, 5-ethenyl-20-deoxyuridine; DAPI, 4’,6-diamidino-2-phenylindole.

Figure 7

(A) Compound 8 induced apoptosis associated with ROS production. The levels of ROS were measured by DCF fluorescence. Data are expressed as means ± standard deviation from three independent experiments (**P<0.01). (B) Flow cytometric analysis of cells stained with Annexin V-FITC/PI after treatment with 10 μM Compound 8 combined with hypoxia, or hypoxia only. (C) Statistical results of apoptosis assays. Data are expressed as means ± standard deviation from three independent experiments (**P<0.01). Abbreviations: PI, propidium iodide; FITC, fluorescein isothiocyanate; ROS, reactive oxygen species; DCF, 2′,7′-Dichlorofluorescein.

Figure 8

Compound 8 suppressed tumor growth in the subcutaneous A549 tumor model. Notes: (A) Tumor growth curve in each group. (B) Body weight of experimental animals in each group.