title: A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A.
abstract: Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.
authors.affiliation: Cylacel Ltd, James Lindsay Place, Dundee, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Emery
authors.forename: David A
authors.initials: DA
authors.lastname: Sorrell
authors.forename: Stacy
authors.initials: S
authors.lastname: Lawrence
authors.forename: Emma
authors.initials: E
authors.lastname: Easthope
authors.forename: Mark
authors.initials: M
authors.lastname: Stockdale
authors.forename: David O
authors.initials: DO
authors.lastname: Jones
authors.forename: Daniella
authors.initials: D
authors.lastname: Zheleva
authors.forename: Frederic
authors.initials: F
authors.lastname: Scaerou
authors.forename: David M
authors.initials: DM
authors.lastname: Glover
mesh_terms: D045163:Antibodies, Phospho-Specific / Q000032:analysis* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D064127:Aurora Kinases; D057205:Automation, Laboratory; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D057054:Molecular Imaging; D010755:Organophosphates / Q000494:pharmacology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology; D054852:Small Molecule Libraries; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines
chemical_list: D045163:Antibodies, Phospho-Specific; D000970:Antineoplastic Agents; D064127:Aurora Kinases; D008869:Microtubule-Associated Proteins; D010755:Organophosphates; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011799:Quinazolines; D054852:Small Molecule Libraries; D025521:Tumor Suppressor Proteins; C520647:2-((3-((4-((5-(2-((3-fluorophenyl)amino)-2-oxoethyl)-1H-pyrazol-3-yl)amino)quinazolin-7-yl)oxy)propyl)(ethyl)amino)ethyl dihydrogen phosphate; C120551:TACC3 protein, human; C484810:tozasertib; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines; C406449:LATS2 protein, human