Inhibition of Inositol Polyphosphate Kinases by Quercetin and Related Flavonoids: A Structure-Activity Analysis

Abstract

Dietary flavonoids inhibit certain protein kinases and phospholipid kinases by competing for their ATP-binding sites. These nucleotide pockets have structural elements that are well-conserved in two human small-molecule kinases, inositol hexakisphosphate kinase (IP6K) and inositol polyphosphate multikinase (IPMK), which synthesize multifunctional inositol phosphate cell signals. Herein, we demonstrate that both kinases are inhibited by quercetin and 16 related flavonoids; IP6K is the preferred target. Relative inhibitory activities were rationalized by X-ray analysis of kinase/flavonoid crystal structures; this detailed structure-activity analysis revealed hydrophobic and polar ligand/protein interactions, the degree of flexibility of key amino acid side chains, and the importance of water molecules. The seven most potent IP6K inhibitors were incubated with intact HCT116 cells at concentrations of 2.5 μM; diosmetin was the most selective and effective IP6K inhibitor (>70% reduction in activity). Our data can instruct on pharmacophore properties to assist the future development of inositol phosphate kinase inhibitors. Finally, we propose that dietary flavonoids may inhibit IP6K activity in cells that line the gastrointestinal tract.

Fig. 1.. The roles of IPMK and IP6K in the mammalian InsP metabolic pathway.

‘Ins’ refers to inositol; the subscripts denote the total number of phosphates (‘P’), and the numbers in parentheses describe the positions of the phosphate groups around the inositol ring. Arrows depict metabolic steps, catalyzed by the following enzymes: IP3K, inositol trisphosphate 3-kinase; IPMK, inositol polyphosphate multikinase; INPP5, inositol polyphosphate 5-phosphatase; ITPK1, inositol trisphosphate 6-kinase/ inositol tetrakisphosphate 1-kinase; IP5K, inositol pentakisphosphate 2-kinase; IP6K, inositol hexakisphosphate kinase. The enzymes that are the focus of this study – IPMK and IP6K - are highlighted in red font.

Fig. 2.. Chemical structures of the flavonoids used in this study

.

Fig. 3.. Structure of the hIPMK/2 crystal complex.

A, Surface representation of quercetin binding. The C- and N-lobes are depicted in yellow and orange, respectively; the hinge region (E131 to K139 ) is colored purple, and 2 is shown as a dark green stick model, with the oxygen atoms illustrated in red. All color coding is retained for structural elements shown in the other panels. B, Polar interactions of 2 with hIPMK; 2Fo-Fc electron density maps (skyblue mesh) are contoured at 1.0 σ. Hydrogen bonds are shown in broken gray lines. Water molecules that directly interact with 2 are depicted as red spheres. C, Ribbon plot of hIPMK showing superimposition of bound 2 (this study, PDB = 6M89) and ADP (from , PDB = 5W2H; dark gray stick model; nitrogen atoms are shown in blue). D-G show ligplots and relative spatial positioning of all residues which make either Van der Waals or polar interactions with either 2 (D,E) or ADP (F,G).

Fig. 4.. Variations in the relative spatial positioning of 2 in various kinases.

A, Ribbon plot of a homology model of hIP6K2 into which 2 is docked; color coding is as in Fig. 2. B-E, by using the conserved nucleotide binding residues such as hinge region, metal binding residues and invariant lysine, the pose of 2 in the hIPMK crystal complex (green stick model) is superimposed upon the configuration of 2 (light gray stick model; oxygen atoms are shown in red; broken dark gray lines depict polar contacts) for the following kinase/2 crystal complexes: B, HCK (PDB = 2HCK); C, PIM1 (PDB = 2O3P); D, (PDB = 5AUW); E, PI3K (PDB = 1E8W).

Fig. 5. Crystal structure of hIPMK in complex with various flavonoids.

A-F depict the data obtained from X-ray crystallographic analysis of selected flavonoids soaked into the crystal structure of hIPMK. A 2Fo-Fc map of each bound compound is shown (skyblue mesh), contoured at 1σ. Broken lines depict hydrogen-bond interactions with associated water molecules and key residues. To highlight a rotamer switch, the groups attached to the 3’-carbon on the phenyl ring are annotated for compounds 1 and 6 (panels A and C, respectively).

Fig. 6.. HPLC analysis of the time-dependent effects of selected flavonoids (2, 3 and 7) upon InsP₅, InsP₆ and InsP₇ in HCT116 cells.

A, Representative HPLC analysis of cell extracts prepared from [³H]inositol-labeled HCT116 cells incubated for 3.5 h with either DMSO vehicle (open circles) or 10 μM 2 (blue circles). The total DPM for each peak is: control, InsP₅ = 14677; InsP₆ = 36,327; InsP₇ = 2160; treated with 2, InsP₅ = 10,989; InsP₆ = 39521; InsP₇ = 195. The inset shows the relative levels of InsP₇ in cells treated for 3.5 h with either the indicated concentration of 2 (blue circles) or corresponding DMSO vehicle controls (open circles). B-D, HCT116 cells were treated with 2.5 µM of either 2 (data colored blue), 3 (pink) or 7 (red) for the indicated times. Levels of the following InsPs were assayed by HPLC: B, InsP₇, C, InsP₅, D, InsP₆. Data are means ± standard errors (n=3–4).

Fig. 7.. The effects of flavonoids upon the InsP profile in HCT116 cells.

A-C Levels of the indicated InsPs in HCT116 cells were determined by HPLC as described in the legend to Fig. 6A, following 3.5 h treatment with 2.5 µM of the indicated flavonoids. A, InsP₅, B, InsP₆, C, InsP₇. Data are means ± standard errors (n=3–4); data for 2,3 and 7 are included from Fig. 5 for comparative purposes (using the same color-coding). *, p < 0.05; **, p < 0.02; ***, p < 0.001, all versus controls.

Fig. 8.. The effects of flavonoids upon the InsP profile in HEK293 cells.

A-C Levels of the indicated InsPs in HCT116 cells were determined by HPLC as described in the legend to Fig. 6A, following 3.5 h treatment with 2.5 µM of the indicated flavonoids. A, InsP₅, B, InsP₆, C, InsP₇. Data are means ± standard errors (n=3); *p<0.05 versus corresponding control values.

Fig 9.. The effects of flavonoids and TNP upon AKT activity in HCT116 cells and 3T3-L1 fibroblasts.

Western analysis of AKT activity (T308 phosphorylation) and total AKT in A, HCT116 cells and B, 3T3-L1 pre-adipocytes, in each case following cell treatment for 3 h with either DMSO vehicle control (C), TNP, or 2.5 µM of either 2, 3, or 7. The upper panels depict means ± standard errors (A, n=5; B, n=3) as a ratio of p-AKT (phosphorylated AKT; T308), versus total AKT. The lower panels provide representative Western blots. Color-coding facilitates comparisons with the data in Figs 6 and 7. *p < 0.01; ** p < 0.001, vs control.