title: ATP-mediated kinome selectivity: the missing link in understanding the contribution of individual JAK Kinase isoforms to cellular signaling.
abstract: Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP Km of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or Km ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than Km ATP may provide crucial insights into a compound's activity and selectivity in cellular contexts.
authors.affiliation: Pfizer Worldwide Research , 200 Cambridgepark Drive, Cambridge, Massachusetts 02140, United States.
authors.forename: Atli
authors.initials: A
authors.lastname: Thorarensen
authors.forename: Mary Ellen
authors.initials: ME
authors.lastname: Banker
authors.forename: Andrew
authors.initials: A
authors.lastname: Fensome
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Telliez
authors.forename: Brian
authors.initials: B
authors.lastname: Juba
authors.forename: Fabien
authors.initials: F
authors.lastname: Vincent
authors.forename: Robert M
authors.initials: RM
authors.lastname: Czerwinski
authors.forename: Agustin
authors.initials: A
authors.lastname: Casimiro-Garcia
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D006801:Humans; D053613:Janus Kinase 1 / Q000378:metabolism; D053616:Janus Kinase 3 / Q000378:metabolism; D053612:Janus Kinases / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D050799:STAT5 Transcription Factor; D000255:Adenosine Triphosphate; D053613:Janus Kinase 1; D053616:Janus Kinase 3; D053612:Janus Kinases