The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: A proof-of-concept study

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. [Display omitted] ► Substrate-Activity-Screening is applied to a tyrosine kinase. ► The substrate efficiency of a tetrapeptide was optimized using a modular approach. ► Substrates were then converted into inhibitors by replacement of the tyrosine. ► A correlation between substrate SAR and inhibitor SAR was confirmed. ► We obtained a low single-digit micromolar substrate-site inhibitor of IGF-1R.