title: Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.
abstract: Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.
authors.affiliation: Amgen, Inc., Cambridge, MA 02142, USA.
authors.forename: Allison E
authors.initials: AE
authors.lastname: Drew
authors.forename: Samer
authors.initials: S
authors.lastname: Al-Assaad
authors.forename: Violeta
authors.initials: V
authors.lastname: Yu
authors.forename: Paul
authors.initials: P
authors.lastname: Andrews
authors.forename: Patricia
authors.initials: P
authors.lastname: Merkel
authors.forename: Stephen
authors.initials: S
authors.lastname: Szilvassy
authors.forename: Renee
authors.initials: R
authors.lastname: Emkey
authors.forename: Richard
authors.initials: R
authors.lastname: Lewis
authors.forename: Rachael L
authors.initials: RL
authors.lastname: Brake
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D001681:Biological Assay; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D000077547:Crizotinib; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism*; D010880:Piperidines / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011720:Pyrazoles; D011725:Pyridines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases
chemical_list: D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D010880:Piperidines; D011720:Pyrazoles; D011725:Pyridines; D000077547:Crizotinib; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases