4 results on '"Dmitri Kireev"'
Search Results
2. A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors.
- Author
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Brandi M Baughman, Huanchen Wang, Yi An, Dmitri Kireev, Michael A Stashko, Henning J Jessen, Kenneth H Pearce, Stephen V Frye, and Stephen B Shears
- Subjects
Medicine ,Science - 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.
- Published
- 2016
- Full Text
- View/download PDF
3. Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε.
- Author
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Jessica E Hutti, Melissa A Porter, Adam W Cheely, Lewis C Cantley, Xiaodong Wang, Dmitri Kireev, Albert S Baldwin, and William P Janzen
- Subjects
Medicine ,Science - Abstract
IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.
- Published
- 2012
- Full Text
- View/download PDF
4. Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε
- Author
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William P. Janzen, Albert S. Baldwin, Adam W. Cheely, Lewis C. Cantley, Xiaodong Wang, Melissa A. Porter, Dmitri Kireev, and Jessica E. Hutti
- Subjects
cells ,Amino Acid Motifs ,lcsh:Medicine ,environment and public health ,Biochemistry ,Substrate Specificity ,0302 clinical medicine ,Drug Discovery ,Molecular Cell Biology ,Phosphorylation ,skin and connective tissue diseases ,lcsh:Science ,0303 health sciences ,Multidisciplinary ,Protein Kinase Signaling Cascade ,Kinase ,Drug discovery ,Small molecule ,Innate Immunity ,Signaling Cascades ,3. Good health ,I-kappa B Kinase ,030220 oncology & carcinogenesis ,Medicine ,biological phenomena, cell phenomena, and immunity ,Signal transduction ,Research Article ,Signal Transduction ,Test Evaluation ,High-throughput screening ,Molecular Sequence Data ,Immunology ,Biology ,Protein Serine-Threonine Kinases ,Small Molecule Libraries ,03 medical and health sciences ,Peptide Library ,Diagnostic Medicine ,Humans ,Amino Acid Sequence ,Peptide library ,Protein Kinase Inhibitors ,030304 developmental biology ,Enzyme Assays ,Inflammation ,Protein-Serine-Threonine Kinases ,lcsh:R ,Immunity ,Peptide Fragments ,High-Throughput Screening Assays ,enzymes and coenzymes (carbohydrates) ,Kinetics ,HEK293 Cells ,Small Molecules ,lcsh:Q - Abstract
IKKe and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKe has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKe. This information enabled the design of an optimal TBK1/IKKe substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKe. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKe. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKe inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.
- Published
- 2012
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