Potent Biological Activity of Fluorinated Derivatives of 2-Deoxy-d-Glucose in a Glioblastoma Model

Abstract

Background: One defining feature of various aggressive cancers, including glioblastoma multiforme (GBM), is glycolysis upregulation, making its inhibition a promising therapeutic approach. One promising compound is 2-deoxy-d-glucose (2-DG), a d-glucose analog with high clinical potential due to its ability to inhibit glycolysis. Upon uptake, 2-DG is phosphorylated by hexokinase to 2-DG-6-phosphate, which inhibits hexokinase and downstream glycolytic enzymes. Unfortunately, therapeutic use of 2-DG is limited by poor pharmacokinetics, suppressing its efficacy.

Methods: To address these issues, we synthesized novel halogenated 2-DG analogs (2-FG, 2,2-diFG, 2-CG, and 2-BG) and evaluated their glycolytic inhibition in GBM cells. Our in vitro and computational studies suggest that these derivatives modulate hexokinase activity differently.

Results: Fluorinated compounds show the most potent cytotoxic effects, indicated by the lowest IC₅₀ values. These effects were more pronounced in hypoxic conditions. ¹⁹F NMR experiments and molecular docking confirmed that fluorinated derivatives bind hexokinase comparably to glucose. Enzymatic assays demonstrated that all halogenated derivatives are more effective HKII inhibitors than 2-DG, particularly through their 6-phosphates. By modifying the C-2 position with halogens, these compounds may overcome the poor pharmacokinetics of 2-DG. The modifications seem to enhance the stability and uptake of the compounds, making them effective at lower doses and over prolonged periods.

Conclusions: This research has the potential to reshape the treatment landscape for GBM and possibly other cancers by offering a more targeted, effective, and metabolically focused therapeutic approach. The application of halogenated 2-DG analogs represents a promising advancement in cancer metabolism-targeted therapies, with the potential to overcome current treatment limitations.

Keywords: 2-deoxy-d-glucose; NMR spectroscopy; cytotoxic action; glycolysis; glycolysis inhibition; halogenated derivatives; hexokinase activity; molecular docking.

Figure 1

Chemical structures of d-glucose, 2-DG, and its halogenated derivatives: 2-FG, 2,2-diFG, 2-BG, 2-CG.

Figure 2

Viability of U-87 and U-251 cells after 72 h treatment with various concentrations of (A) 2-DG [0.5–20 mM] and its halogen derivatives: (B) 2-FG [1–10 mM], (C) 2,2-diFG [0.5–15 mM]. Protein synthesis inhibitor CHX [20 μM] was used as a positive cytotoxic control. Significant differences between the treatment and control means are indicated by *** p < 0.001.

Figure 3

Proliferation of U-87 and U-251 cells after 72 h treatment with various concentrations of (A) 2-DG [2.5–20 mM] and its halogen derivatives: (B) 2-FG [1–5 mM], (C) 2,2-diFG [1–10 mM]. Protein synthesis inhibitor CHX [20 μM] was used as a positive cytotoxic control. Significant differences between the treatment and control means are indicated by *** p < 0.001.

Figure 4

Protein synthesis of U-87 and U-251 cells after 72 h treatment with various concentrations of (A) 2-DG [2.5–10 mM] and its halogen derivatives: (B) 2-FG [1–5 mM], (C) 2,2-diFG [1–10 mM]. Protein synthesis inhibitor CHX [20 μM] was used as a positive cytotoxic control. Significant differences between the treatment and control means are indicated by *** p < 0.001.

Figure 5

Viability of U-87 and U-251 cells after 72 h treatment with various concentrations of (A) 2-DG [0.5–20 mM] and its halogen derivatives: (B) 2-FG [1–10 mM], (C) 2,2-diFG [0.5–15 mM] in normoxia and hypoxia-like (DMOG + Rho) conditions. Significant differences between the treatment and control means are indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, ns—no statistical significance.

Figure 6

Intracellular (cells) and extracellular (medium) lactate production of U-251 and U-87 cells after 72 h treatment with various concentrations of (A) 2-DG [2.5–10 mM] and its halogen derivatives: (B) 2-FG [1–5 mM], (C) 2,2-diFG [0.5–5 mM]. Significant differences between the treatment and control means are indicated by *** p < 0.001.

Figure 7

Viability of U-87 cells after 72 h combined treatment of CQ [10 μM] with IC₅₀ concentrations of 2-DG [5 mM], 2-FG [3 mM], and 2,2-diFG [5 mM]. Significant differences between the treatment and control means are indicated by ** p < 0.01, *** p < 0.001, ns—no statistical significance.

Figure 8

Affinity of halogenated analogs of 2-DG to hexokinase determined by ¹⁹F NMR relaxation experiment. In each case, the K_d was determined separately for α and β anomers, and the corresponding fitting curves are depicted as blue and orange, respectively.

Figure 9

Molecular docking of 2-DG and its derivatives to crystal structure HKII (PDB entry 2NTZ). In each panel, selected hydrogen atoms and amino acid residues of HKII were omitted for clarity, making binding site visible. Typical H···O hydrogen bonds are depicted as pink dotted lines. H-bonds with less stringed geometrical constraints and close contacts involving position 2 in the pyranose ring are depicted as green dotted lines. If not mentioned otherwise only the α anomers are shown. Panels A-F show the docking of following compounds: (A) Glc (experimental data from literature, PDB entry 2NTZ); (B) 2-DG; (C) 2-FG, superposition of both anomers, H···O hydrogen bonds formed by the β anomer are depicted as orange dotted lines, H···F hydrogen bonds are depicted as green dotted lines; (D) 2,2-diFG, superposition of both anomers, H···O hydrogen coloring scheme is as in the panel C, H···F hydrogen bonds are depicted as dotted lines colored green and black for anomers α and β, respectively; (E) 2-CG; (F) 2-BG.

Figure 10

Hirshfeld surface analysis of ligand-protein interactions for selected compounds (A) 2-DG (red—strong O···H H bonds, orange—H···H contacts), (B) 2-FG, anomers α and β are shown on top and bottom, respectively (dark pink—possible weak F···O halogen bond, lime—medium strong F···H H-bonds, (C) 2,2′-diFG, anomers α and β are shown at top and bottom, respectively (red—atomic clash with water molecules (docking artifact), dark pink—possible weak F···O halogen bond), (D) 2-CG (violet—possible weak Cl···O halogen bond, green—weak Cl···H H-bonds, lime—medium strong F···H H-bonds, dark pink—possible weak F···O halogen bond).

Figure 11

Inhibition of HKII by 2-DG and its fluorinated-derivatives (2-FG, 2,2-diFG). The HKII activity is normalized to 1.0 in the absence of any inhibitors.