title: A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors.
abstract: 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.
authors.affiliation: Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.|Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Brandi M
authors.identifier: http://orcid.org/0000-0002-8861-1345
authors.initials: BM
authors.lastname: Baughman
authors.affiliation: Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
authors.forename: Huanchen
authors.initials: H
authors.lastname: Wang
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Yi
authors.initials: Y
authors.lastname: An
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Kireev
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Stashko
authors.affiliation: Institute of Organic Chemistry, Albert-Ludwigs-University of Freiburg, Freiburg 79104, Germany.
authors.forename: Henning J
authors.initials: HJ
authors.lastname: Jessen
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Pearce
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Frye
authors.affiliation: Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D011994:Recombinant Proteins; C402123:inositol heptakisphosphate; D000255:Adenosine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C519580:PPIP5K1 protein, human