title: A FLIPR-based assay to assess potency and selectivity of inhibitors of the TEC family kinases Btk and Itk.
abstract: Bruton's tyrosine kinase (Btk) and interleukin-2-inducible T cell kinase (Itk) are members of the TEC family of nonreceptor tyrosine kinases and are expressed primarily in B and T cells, respectively. Both kinases are critically involved in lymphocyte development and signal transduction. In particular, Btk and Itk regulate calcium mobilization subsequent to antigen receptor stimulation. Small molecule antagonists that specifically inhibit either Btk or Itk may allow for selective modulation of B cell or T cell activity and may be useful in treating inflammatory and autoimmune conditions. We have developed a medium-throughput fluorescent imaging plate reader (FLIPR)- based calcium flux assay that can be used to assay potential Btk and Itk inhibitors. This assay takes advantage of Btk-deficient DT40 (DT40-Btk-/-) chicken B cells, which are unable to mobilize calcium in response to cross-linking of their B cell receptor (BCR). Ectopic expression of TEC family kinases can restore antigen receptor signaling in these cells. We have generated stable DT40-Btk-/- lines expressing either wild-type human Btk (huBtk) or a chimeric Btk-Itk kinase (huBtk-Itk) molecule-a Btk protein whose kinase domain has been replaced by the kinase domain of Itk. Expression of either huBtk or huBtk-Itk in DT40-Btk-/- cells restores calcium flux in response to BCR engagement. Using Btk- and Itk-selective inhibitors, we show that inhibition of calcium responses in huBtk-Itk-DT40-Btk-/- cells and huBtk-DT40-Btk-/- cells is dependent on the Itk or Btk kinase domain, respectively. Thus, the FLIPR assay described here can be used to assess, compare, and rank the potency and selectivity of inhibitors of Itk and Btk kinases.
authors.affiliation: Inflammation, Wyeth Research, Cambridge, MA 02140, USA. jdouhan@wyeth.com
authors.forename: John
authors.initials: J
authors.lastname: Douhan
authors.forename: Joy S
authors.initials: JS
authors.lastname: Miyashiro
authors.forename: Xiaochuan
authors.initials: X
authors.lastname: Zhou
authors.forename: Derek C
authors.initials: DC
authors.lastname: Cole
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wu
authors.forename: Mary
authors.initials: M
authors.lastname: Collins
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Dunussi-Joannopoulos
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*; D005456:Fluorescent Dyes; D005470:Fluorometry; D005612:Freeze Drying; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting; D007834:Lasers; D030781:Organisms, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C080813:emt protein-tyrosine kinase; D002118:Calcium