title: Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.
abstract: Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.
authors.affiliation: Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois 60064, United States. kenneth.m.comess@abbott.com
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Comess
authors.forename: Chaohong
authors.initials: C
authors.lastname: Sun
authors.forename: Cele
authors.initials: C
authors.lastname: Abad-Zapatero
authors.forename: Eric R
authors.initials: ER
authors.lastname: Goedken
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Gum
authors.forename: David W
authors.initials: DW
authors.lastname: Borhani
authors.forename: Maria
authors.initials: M
authors.lastname: Argiriadi
authors.forename: Duncan R
authors.initials: DR
authors.lastname: Groebe
authors.forename: Yong
authors.initials: Y
authors.lastname: Jia
authors.forename: Jill E
authors.initials: JE
authors.lastname: Clampit
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Haasch
authors.forename: Harriet T
authors.initials: HT
authors.lastname: Smith
authors.forename: Sanyi
authors.initials: S
authors.lastname: Wang
authors.forename: Danying
authors.initials: D
authors.lastname: Song
authors.forename: Michael L
authors.initials: ML
authors.lastname: Coen
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Cloutier
authors.forename: Hua
authors.initials: H
authors.lastname: Tang
authors.forename: Xueheng
authors.initials: X
authors.lastname: Cheng
authors.forename: Christopher
authors.initials: C
authors.lastname: Quinn
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
authors.forename: Zhili
authors.initials: Z
authors.lastname: Xin
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Fry
authors.forename: Vincent
authors.initials: V
authors.lastname: Stoll
authors.forename: Teresa I
authors.initials: TI
authors.lastname: Ng
authors.forename: David
authors.initials: D
authors.lastname: Banach
authors.forename: Doug
authors.initials: D
authors.lastname: Marcotte
authors.forename: David J
authors.initials: DJ
authors.lastname: Burns
authors.forename: David J
authors.initials: DJ
authors.lastname: Calderwood
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Hajduk
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D057166:High-Throughput Screening Assays; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D000255:Adenosine Triphosphate; D048055:Mitogen-Activated Protein Kinase 8; D048051:p38 Mitogen-Activated Protein Kinases