title: Development of phosphocellulose paper-based screening of inhibitors of lipid kinases: case study with PI3Kβ.
abstract: The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transduction pathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of these pathways are common in oncogenesis, and they are known to be altered in other metabolic disorders as well. Despite its huge potential as an attractive target in these diseases, there is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhibitors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinase screening platform using a phosphocellulose plate that involves transfer of radiolabeled [γ-(32)P]ATP to phosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phosphocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies toward PI3K isoforms. ATP and lipid apparent Km for both were determined and IC50 values generated that matched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay (PI3Kβ as the target) as an excellent platform for the identification of novel chemical entities in PI3K drug discovery.
authors.affiliation: Biology Division, GVK Biosciences Pvt. Ltd., Hyderabad 500076, Andhra Pradesh, India; Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, 500085 Hyderabad, Andhra Pradesh, India.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Yanamandra
authors.affiliation: VINS BIO, Kothur Mandal, Mahaboobnagar District 509325, Andhra Pradesh, India.
authors.forename: Labanyamoy
authors.initials: L
authors.lastname: Kole
authors.affiliation: Centre for Biotechnology, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, 500085 Hyderabad, Andhra Pradesh, India.
authors.forename: Archana
authors.initials: A
authors.lastname: Giri
authors.affiliation: Biology Division, GVK Biosciences Pvt. Ltd., Hyderabad 500076, Andhra Pradesh, India. Electronic address: sayan.mitra@gvkbio.com.
authors.forename: Sayan
authors.initials: S
authors.lastname: Mitra
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation; D057075:Enzyme Assays / Q000295:instrumentation*; D004867:Equipment Design; D006801:Humans; D010209:Paper; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D002482:Cellulose; C008881:phosphocellulose; D019869:Phosphatidylinositol 3-Kinases
keywords: IC(50); Lipid; Phosphatidyl inositol 3-kinase; Phosphocellulose; PtdIns; PtdSer