دورية أكاديمية
Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors.
| العنوان: | Development of High-Throughput Assays for Evaluation of Hematopoietic Progenitor Kinase 1 Inhibitors. |
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| المؤلفون: | Lacey BM; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Xu Z; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Chai X; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Laskey J; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Fradera X; Department of Computational and Structural Chemistry, Merck & Co., Inc., Boston, MA, USA., Mittal P; Department of Oncology Early Discovery, Merck & Co., Inc., Boston, MA, USA., Mishra S; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Piesvaux J; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Saradjian P; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Shaffer L; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Vassileva G; Department of Genetics and Pharmacogenomics, Merck & Co., Inc., Boston, MA, USA., Gerdt C; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Wang Y; Department of Oncology Early Discovery, Merck & Co., Inc., Boston, MA, USA., Ferguson H; Department of Preclinical Development, Merck & Co., Inc., Boston, MA, USA., Smith DM; Department of PPDM, Merck & Co., Inc., Boston, MA, USA., Ballard J; Department of PPDM, Merck & Co., Inc., Boston, MA, USA., Wells S; Department of Oncology Early Discovery, Merck & Co., Inc., Boston, MA, USA., Jain R; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Mueller U; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Addona G; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Kariv I; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Methot JL; Department of Discovery Chemistry, Merck & Co., Inc., Boston, MA, USA., Bittinger M; Department of Oncology Early Discovery, Merck & Co., Inc., Boston, MA, USA., Ranganath S; Department of Oncology Early Discovery, Merck & Co., Inc., Boston, MA, USA., Mcleod R; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Pasternak A; Department of Discovery Chemistry, Merck & Co., Inc., Boston, MA, USA., Miller JR; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA., Xu H; Department of Quantitative Biosciences, Merck & Co., Inc., Boston, MA, USA. |
| المصدر: | SLAS discovery : advancing life sciences R & D [SLAS Discov] 2021 Jan; Vol. 26 (1), pp. 88-99. Date of Electronic Publication: 2020 Aug 26. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: SAGE Publications Country of Publication: United States NLM ID: 101697563 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2472-5560 (Electronic) Linking ISSN: 24725552 NLM ISO Abbreviation: SLAS Discov Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Thousand Oaks, CA : SAGE Publications, [2017]- |
| مواضيع طبية MeSH: | Drug Discovery/*methods , High-Throughput Screening Assays/*methods , Protein Kinase Inhibitors/*chemistry , Protein Kinase Inhibitors/*pharmacology , Protein Serine-Threonine Kinases/*antagonists & inhibitors , Protein Serine-Threonine Kinases/*chemistry, Animals ; Cell Line ; Humans ; Leukocytes, Mononuclear/drug effects ; Leukocytes, Mononuclear/metabolism ; Mice ; T-Lymphocytes/drug effects ; T-Lymphocytes/metabolism |
| مستخلص: | Hematopoietic progenitor kinase 1 (HPK1), also referred to as mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1), is a serine/threonine kinase that negatively regulates T-cell signaling by phosphorylating Ser376 of Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76), a critical mediator of T-cell receptor activation. HPK1 loss of function mouse models demonstrated enhanced immune cell activation and beneficial antitumor activity. To enable discovery and functional characterization of high-affinity small-molecule HPK1 inhibitors, we have established high-throughput biochemical, cell-based, and novel pharmacodynamic (PD) assays. Kinase activity-based time-resolved fluorescence energy transfer (TR-FRET) assays were established as the primary biochemical approach to screen for potent inhibitors and assess selectivity against members of MAP4K and other closely related kinases. A proximal target engagement (TE) assay quantifying pSLP-76 levels as a readout and a distal assay measuring IL-2 secretion as a functional response were established using human peripheral blood mononuclear cells (PBMCs) from two healthy donors. Significant correlations between biochemical and cellular assays as well as excellent correlation between the two donors for the cellular assays were observed. pSLP-76 levels were further used as a PD marker in the preclinical murine model. This effort required the development of a novel ultrasensitive single-molecule array (SiMoA) assay to monitor pSLP-76 changes in mouse spleen. |
| فهرسة مساهمة: | Keywords: HPK1; IL-2; SLP-76; SiMoA; pSLP-76 |
| المشرفين على المادة: | 0 (Protein Kinase Inhibitors) EC 2.7.1.11 (hematopoietic progenitor kinase 1) EC 2.7.11.1 (Protein Serine-Threonine Kinases) |
| تواريخ الأحداث: | Date Created: 20200827 Date Completed: 20220217 Latest Revision: 20220830 |
| رمز التحديث: | 20231215 |
| DOI: | 10.1177/2472555220952071 |
| PMID: | 32844715 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 2472-5560 |
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| DOI: | 10.1177/2472555220952071 |