title: The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: a proof-of-concept study.
abstract: Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although "traditional" kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. In order to facilitate the identification of potential chemical starting points for substrate-site inhibition approaches, we desired to investigate the application of Substrate Activity Screening to kinases. We herein report a proof-of-concept study demonstrating, on a model tyrosine kinase, that the key requirements of this methodology can be met. Namely, using peptides as model substrates, we show that a simple ADP-accumulation assay can be used to monitor substrate efficiency and that efficiency can be optimized in a modular manner. More importantly, we demonstrate that structure-efficiency relationships translate into structure-activity relationships upon conversion of the substrates into inhibitors.
authors.affiliation: Novartis Institutes for BioMedical Research, Novartis Campus, CH-4002 Basel, Switzerland.
authors.forename: Julien
authors.initials: J
authors.lastname: Chapelat
authors.forename: Frédéric
authors.initials: F
authors.lastname: Berst
authors.forename: Andreas L
authors.initials: AL
authors.lastname: Marzinzik
authors.forename: Henrik
authors.initials: H
authors.lastname: Moebitz
authors.forename: Peter
authors.initials: P
authors.lastname: Drueckes
authors.forename: Joerg
authors.initials: J
authors.lastname: Trappe
authors.forename: Doriano
authors.initials: D
authors.lastname: Fabbro
authors.forename: Dieter
authors.initials: D
authors.lastname: Seebach
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D020794:Receptor Protein-Tyrosine Kinases