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

作者：Baughman Brandi M; Wang Huanchen; An Yi; Kireev Dmitri; Stashko Michael A; Jessen Henning J; Pearce Kenneth H; Frye Stephen V; Shears Stephen B

来源：PLos One, 2016, 11(10): e0164378.

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 'highenergy' 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 mu M, 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 mu M) and UNC10225498 (Kd = 1.37 +/- 0.03 mu M). These Kd values lie within the 1 +/- 10 mu M 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 mu M, 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.

• 出版日期2016-10-13