ABSTRACT
We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.
Subject(s)
Antineoplastic Agents/pharmacology , Fibroblast Growth Factors/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Design , Drug Evaluation, Preclinical , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Receptors, Fibroblast Growth Factor/genetics , Signal Transduction/drug effects , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
This chapter describes nuclear magnetic resonance (NMR) methods that can be used to determine the structures of protein complexes. Many of these techniques are also applicable to other systems (e.g., protein-nucleic acid complexes). In the first section, we discuss methodologies for optimizing the sample conditions for the study of complexes. This is followed by a description of the methods that can be used to map interfaces when a full structure determination of the complex is not appropriate or not possible. We then describe experimental approaches for resonance assignment in complexes, these are essentially the same as those for isolated proteins. Subheading 6. describes the different types of so-called X-filtered NMR experiments that have been devised to separate and selectively observe either inter- or intramolecular structural information. These filtered NMR experiments are then exploited in the experimental strategies for structure determination of either protein complexes or homodimeric proteins. This is followed by a description of the calculation of their structures. Finally, we present case studies from three projects carried out in our laboratory, where we successfully used the methods presented in this chapter.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/chemistry , Animals , GTP-Binding Proteins/chemistry , Histones/chemistry , Macromolecular Substances , Mice , Models, Molecular , Oligonucleotides/chemistry , Peptides/chemistry , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Protein Structure, Tertiary , p21-Activated KinasesABSTRACT
HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.
