A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

• Published in: PloS one Vol. 11; no. 10; p. e0164378
• Main Authors: Baughman, Brandi M, Wang, Huanchen, An, Yi, Kireev, Dmitri, Stashko, Michael A, Jessen, Henning J, Pearce, Kenneth H, Frye, Stephen V, Shears, Stephen B
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.10.2016; Public Library of Science (PLoS)
• Subjects: Adenosine diphosphate; Adenosine triphosphate; Adenosine Triphosphate - metabolism; ATP; Binding Sites; Biocatalysis; Biological activity; Biology; Biology and Life Sciences; Calorimetry; Cascades; Chromatography, High Pressure Liquid; Cluster analysis; Docking; Energy metabolism; Energy transfer; Environmental health; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Enzymes; Fluorescence; Fluorescence resonance energy transfer; Health sciences; High-performance liquid chromatography; High-throughput screening; High-Throughput Screening Assays; Humans; Inhibition; Inositol; Inositol phosphate; Inositol Phosphates - metabolism; Kinases; Kinetics; Laboratories; Ligands; Liquid chromatography; Medical screening; Metabolism; Molecular Docking Simulation; Pharmacology; Phosphates; Phosphorylation; Phosphotransferases (Phosphate Group Acceptor) - antagonists & inhibitors; Phosphotransferases (Phosphate Group Acceptor) - genetics; Phosphotransferases (Phosphate Group Acceptor) - metabolism; Physical Sciences; Physiological aspects; Protein Binding; Protein kinase; Protein kinases; Protein Structure, Tertiary; Proteins; Pyrophosphates; R&D; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Research & development; Research and Analysis Methods; Screening; Signal transduction; Signaling; Strategy; Substrate Specificity; Transduction; North Carolina; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0164378

Pharmacological tools-'chemical probes'-that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes 'high-energy' inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z' = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known-or conjectured based on their structures-to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1-10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.