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

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.

Experimental and Computational Studies on Structure and Energetic Properties of Halogen Derivatives of 2-Deoxy-D-Glucose.

The results of structural studies on a series of halogen-substituted derivatives of 2-deoxy-D-glucose (2-DG) are reported. 2-DG is an inhibitor of glycolysis, a metabolic pathway crucial for cancer cell proliferation and viral replication in host cells, and interferes with D-glucose and D-mannose metabolism. Thus, 2-DG and its derivatives are considered as potential anticancer and antiviral drugs. X-ray crystallography shows that a halogen atom present at the C2 position in the pyranose ring does not significantly affect its conformation. However, it has a noticeable effect on the crystal structure. Fluorine derivatives exist as a dense 3D framework isostructural with the parent compound, while Cl- and I-derivatives form layered structures. Analysis of the Hirshfeld surface shows formation of hydrogen bonds involving the halogen, yet no indication for the existence of halogen bonds. Density functional theory (DFT) periodic calculations of cohesive and interaction energies (at the B3LYP level of theory) have supported these findings. NMR studies in the solution show that most of the compounds do not display significant differences in their anomeric equilibria, and that pyranose ring puckering is similar to the crystalline state. For 2-deoxy-2-fluoro-D-glucose (2-FG), electrostatic interaction energies between the ligand and protein for several existing structures of pyranose 2-oxidase were also computed. These interactions mostly involve acidic residues of the protein; single amino-acid substitutions have only a minor impact on binding. These studies provide a better understanding of the structural chemistry of halogen-substituted carbohydrates as well as their intermolecular interactions with proteins determining their distinct biological activity.

A novel high-dimensional NMR experiment for resolving protein backbone dihedral angle ambiguities.

Intrinsically disordered proteins (IDPs) are challenging established structural biology perception and urge a reassessment of the conventional understanding of the subtle interplay between protein structure and dynamics. Due to their importance in eukaryotic life and central role in protein interaction networks, IDP research is a fascinating and highly relevant research area in which NMR spectroscopy is destined to be a key player. The flexible nature of IDPs, as a result of the sampling of a vast conformational space, however, poses a tremendous scientific challenge, both technically and theoretically. Pronounced signal averaging results in narrow signal dispersion and requires higher dimensionality NMR techniques. Moreover, a fundamental problem in the structural characterization of IDPs is the definition of the conformational ensemble sampled by the polypeptide chain in solution, where often the interpretation relies on the concept of 'residual structure' or 'conformational preference'. An important source of structural information is information-rich NMR experiments that probe protein backbone dihedral angles in a unique manner. Cross-correlated relaxation experiments have proven to fulfil this task as they provide unique information about protein backbones, particularly in IDPs. Here we present a novel cross-correlation experiment that utilizes non-uniform sampling detection schemes to resolve protein backbone dihedral ambiguities in IDPs. The sensitivity of this novel technique is illustrated with an application to the prototypical IDP [Formula: see text]-Synculein for which unexpected deviations from random-coil-like behaviour could be observed.

19F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

A new 19F NMR method is presented which can be used to detect weak protein binding of small molecules with up to mM affinity. The method capitalizes on the synthetic availability of unique SF5 containing compounds and the generation of five-quantum coherences (5QC). Given the high sensitivity of 5QC relaxation to exchange events (i.e. reversible protein binding) fragments which bind to the target with weak affinity can be identified. The utility of the method in early stage drug discovery programs is demonstrated with applications to two model proteins, the neurotoxic NGAL and the prominent tumor target ß-catenin.

Protonation-dependent conformational variability of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity and undergo rearrangements of the time-averaged conformational ensemble on changes of environmental conditions (e.g., in ionic strength, pH, molecular crowding). In contrast to stably folded proteins, IDPs often form compact conformations at acidic pH. The biological relevance of this process was, for example, demonstrated by nuclear magnetic resonance studies of the aggregation prone (low pH) state of α-synuclein. In this study, we report a large-scale analysis of the pH dependence of disordered proteins using the recently developed meta-structure approach. The meta-structure analysis of a large set of IDPs revealed a significant tendency of IDPs to form α-helical secondary structure elements and to preferentially fold into more compact structures under acidic conditions. The predictive validity of this novel approach was demonstrated with applications to the tumor-suppressor BASP1 and the transcription factor Tcf4.

(1)H, (13)C, and (15)N chemical shifts assignments for human endothelial monocyte-activating polypeptide EMAP II.

Endothelial and monocyte-activating polypeptide II (EMAP II) is a cytokine that plays an important role in inflammation, apoptosis and angiogenesis processes in tumour tissues. Structurally, the EMAP II is a 169 amino acid residues long C-terminal domain (residues 147-312) of auxiliary tRNA binding protein p43. In spite of existence in pdb databank of two X-ray structures there are some important aspects of EMAP II cytokine function which are still not fully understood in detail. To obtain information about 3D structure and backbone dynamic processes in solution we perform structure evaluation of human EMAP II cytokine by NMR spectroscopy. The standard approach to sequence-specific backbone assignment using 3D NMR data sets was not successful in our studies and was supplemented by recently developed 4D NMR experiments with random sampling of evolution time space. Here we report the backbone and side chain (1)H, (13)C, and (15)N chemical shifts in solution for recombinant EMAP II cytokine together with secondary structure provided by TALOS + software.

¹H, ¹³C and ¹5N resonance assignments of human BASP1.

Brain acid-soluble protein 1 (BASP1, CAP-23, NAP-22) appears to be implicated in diverse cellular processes. An N-terminally myristoylated form of BASP1 has been discovered to participate in the regulation of actin cytoskeleton dynamics in neurons, whereas non-myristoylated nuclear BASP1 acts as co-suppressor of the potent transcription regulator WT1 (Wilms' Tumor suppressor protein 1). Here we report NMR chemical shift assignment of recombinant human BASP1 fused to an N-terminal cleavable His6-tag.