title: Development and application of PI3K assays for novel drug discovery.
abstract: BACKGROUND
  Phosphoinositide 3-kinases (PI3Ks) constitute one of the most important signaling pathways, playing a vital role in cellular differentiation and proliferation with a key function in cellular receptor triggered signal transduction downstream of tyrosine kinase receptors and/or G-protein coupled receptors. PI3K promotes cell survival proliferation, protein synthesis and glucose metabolism by generating secondary messengers phospholipid phosphatidyl 3,4,5-triphosphate and signaling via AKT/mTOR regulation. Deregulation of PI3K pathways have been observed in cancer, diabetes, neurological and inflammatory diseases and is an attractive target for pharmaceutical industries.
  
  
  METHODS
  In this review, the authors explain different PI3K assay methodologies. Furthermore, the authors summarize the techno-scientific principles and their utility in profiling novel chemical entities against PI3Ks. Specifically, the authors compare different PI3K assay formats explaining their mode of detection as well as their advantages and limitations for drug discovery efforts.
  
  
  CONCLUSIONS
  Developing lipid (PI3K) kinase assays involves significant effort and a rational understanding is needed due to the intrinsic lipidic nature of phospholipid phosphatidyl 4,5-biphosphate, which is used as an in vitro substrate for assays with PI3K isoforms. The assay of choice should be versatile, homogenous and definitely adaptable for high-throughput screening campaigns. Additionally, these assays are expected to dissect the mechanism of action of novel compounds (inhibitor characterization) against PI3K. Existing methods provide the versatility to medicinal chemists such that they can choose one or more assay platform to progress their compounds while profiling and/or inhibitor characterization.
authors.affiliation: Scientist, GVK Biosciences Private Ltd, Biology , Campus MLR 1, Survey Nos. 125 (part) and 126, IDA Mallapur, Hyderabad, Telangana, 500076 , India +91 9989086492 ; Mahesh.yanamandra@gvkbio.com.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Yanamandra
authors.forename: Sayan
authors.initials: S
authors.lastname: Mitra
authors.forename: Archana
authors.initials: A
authors.lastname: Giri
mesh_terms: D000818:Animals; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D054852:Small Molecule Libraries
keywords: ATP; LY294002; Wortmannin; drug discovery; fluorescence; homogeneous time-resolved fluorescence assay; inhibitor; kinase assay; luminescence; nitrocellulose; phosphocellulose; phosphoinositide 3-kinases; phospholipid phosphatidyl 3,4,5-triphosphate; phospholipid phosphatidyl 4,5-biphosphate; radioactive; screening; thin layer chromatography