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.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Phosphotransferases (Phosphate Group Acceptor)/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quercetin/pharmacology , Binding Sites , Crystallography, X-Ray , HCT116 Cells , Humans , Inositol Phosphates/metabolism , Molecular Structure , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Quercetin/chemistry , Quercetin/metabolism , Structure-Activity RelationshipABSTRACT
Inositol hexakisphosphate kinases (IP6Ks) regulate a myriad of cellular processes, not only through their catalytic activity (which synthesizes InsP7, a multifunctional inositol pyrophosphate signaling molecule) but also through protein-protein interactions. To further study the enzymatic function and distinguish between these different mechanisms, specific inhibitors that target IP6K catalytic activity are required. Only one IP6K inhibitor is commonly used: N2-( m-(trifluoromethyl)benzyl) N6-( p-nitrobenzyl)purine (TNP). TNP is, however, compromised by weak potency, inability to distinguish between IP6K isoenzymes, off-target activities, and poor pharmacokinetic properties. Herein, we describe a new inhibitor discovery strategy, based on the high degree of structural conservation of the nucleotide-binding sites of IP6Ks and protein kinases; we screened for novel IP6K2 inhibitors using a focused set of compounds with features known, or computationally predicted, to target nucleotide binding by protein kinases. We developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay of adenosine diphosphate (ADP) formation from adenosine triphosphate (ATP). Novel hit compounds for IP6K2 were identified and validated with dose-response curves and an orthogonal assay. None of these inhibitors affected another inositol pyrophosphate kinase, PPIP5K. Our screening strategy offers multiple IP6K2 inhibitors for future development and optimization. This approach will be applicable to inhibitor discovery campaigns for other inositol phosphate kinases.