title: Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor.
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.
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA (UK).|Present address: Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152 Martinsried (Germany).
authors.forename: Leon
authors.initials: L
authors.lastname: Harrington
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, OX3 7DQ (UK).|Present address: Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich (Switzerland).
authors.forename: Leila T
authors.initials: LT
authors.lastname: Alexander
authors.affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, OX3 7DQ (UK).
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA (UK). hagan.bayley@chem.ox.ac.uk.
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
mesh_terms: D055808:Drug Discovery; D015394:Molecular Structure; D058608:Nanopores; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D011494:Protein Kinases / Q000378:metabolism*; D051573:Proto-Oncogene Proteins c-pim-1 / Q000037:antagonists & inhibitors*
chemical_list: D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D051573:Proto-Oncogene Proteins c-pim-1; C406031:proto-oncogene proteins pim
keywords: drug discovery; inhibitors; protein kinases; screening; single-molecule studies