Pim Kinase Inhibitors Evaluated with a Single‐Molecule Engineered Nanopore Sensor |
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| Authors: | Dr. Leon Harrington Dr. Leila T. Alexander Prof. Dr. Stefan Knapp Prof. Dr. Hagan Bayley |
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| Affiliation: | 1. Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA (UK);2. Present address: Max‐Planck‐Institut für Biochemie, Am Klopferspitz 18, 82152 Martinsried (Germany);3. Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, OX3 7DQ (UK);4. Present address: Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich (Switzerland) |
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| Abstract: | Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label‐free determination of inhibition constants. The procedure circumvents many sources of bias or false‐positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips. |
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| Keywords: | drug discovery inhibitors protein kinases screening single‐molecule studies |
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