title: Novel synthesis and structural characterization of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance.
abstract: To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing how the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.
authors.affiliation: Structural Biology and Computational Chemistry, Wyeth Research, 200 CambridgePark Drive, Cambridge, Massachusetts 02140, USA.
authors.forename: Franklin J
authors.initials: FJ
authors.lastname: Moy
authors.forename: Arthur
authors.initials: A
authors.lastname: Lee
authors.forename: Lori Krim
authors.initials: LK
authors.lastname: Gavrin
authors.forename: Zhang Bao
authors.initials: ZB
authors.lastname: Xu
authors.forename: Annette
authors.initials: A
authors.lastname: Sievers
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kieras
authors.forename: Wayne
authors.initials: W
authors.lastname: Stochaj
authors.forename: Lidia
authors.initials: L
authors.lastname: Mosyak
authors.forename: John
authors.initials: J
authors.lastname: McKew
authors.forename: Désirée H H
authors.initials: DH
authors.lastname: Tsao
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D013113:Spin Labels / Q000138:chemical synthesis*
chemical_list: D047428:Protein Kinase Inhibitors; D013113:Spin Labels; D000255:Adenosine Triphosphate; D011494:Protein Kinases