ABSTRACT
The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.
Subject(s)
I-kappa B Kinase/antagonists & inhibitors , Indoles/chemistry , Protein Kinase Inhibitors/chemistry , Animals , Biological Availability , Disease Models, Animal , Half-Life , Humans , I-kappa B Kinase/metabolism , Indoles/chemical synthesis , Indoles/pharmacokinetics , Lung/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all alpha-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity.
Subject(s)
Peptidylprolyl Isomerase/chemistry , Phosphoprotein Phosphatases/chemistry , Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Binding Sites/genetics , Crystallography, X-Ray , Dimerization , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptides/chemistry , Proline/chemistry , Protein Conformation , Protein Phosphatase 2 , Protein Structure, Quaternary , Protein Structure, Secondary , Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.
