Substrate-Competitive Inhibitors of c-Src Kinase.

[摘要] Protein kinases are key mediators of cellular signal transduction and are heavily studied drug targets.Greater than 99% of reported kinase inhibitors act through the same mechanism of competing for binding to the highly conserved ATP pocket.Although ATP-competitive inhibitors have experienced clinical success, the disadvantages associated with them has resulted in significant interest in the discovery of inhibitors that target regions outside of the ATP binding pocket, such as the protein substrate binding site.However, the identification of compounds that can inhibit the kinase-substrate protein-protein interaction has proven challenging, especially for small molecules. To address this problem, we developed screening methodology that can identify small molecule substrate-competitive inhibitors using the tyrosine kinase c-Src as a model system.Studies began with the preparation of a library of peptidic inhibitors to evaluate tyrosine pharmacophores and generate probes for a competitive binding assay.No peptides with potency suitable for probe development were identified, however important structure activity relationships were gleaned for tyrosine pharmacophores.In a second study, a substrate activity screening (SAS) method for tyrosine kinases was developed.Using an assay that monitors ADP production, the first small molecule substrates for any protein kinase were identified.By applying knowledge gained from the previous pharmacophore study, a small molecule substrate (Km = 122 micromolar) was then successfully converted into a substrate-competitive, ATP-noncompetitive inhibitor (Ki = 16 micromolar).The lead inhibitor has improved selectivity compared to an ATP-competitive inhibitor commonly used as a c-Src probe, and has cellular efficacy similar to FDA approved ATP-competitive kinase inhibitors (SK-BR-3 GI50 = 14 micromolar).This SAS method is the only general screening technique for the selective identification of substrate-competitive kinase inhibitors and should be applicable to any tyrosine kinase of interest.In a final study, substrates identified through SAS were applied to the design of nonpeptidic bisubstrate inhibitors.As a whole, the work presented has demonstrated the importance of retaining hydrogen bonding interactions made by the substrate hydroxyl group in order to generate potent inhibitors.Results from this work should advance the discovery of new small molecule substrate-competitive inhibitors through both screening and rational design.

[发布机构] University of Michigan

[效力级别] kinase

[关键词] medicinal chemistry;kinase;substrate-competitive inhibitor;substrate activity screening;Src;Chemistry;Science;Medicinal Chemistry