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1.
PLoS One ; 7(11): e49375, 2012.
Article in English | MEDLINE | ID: mdl-23145167

ABSTRACT

Soluble amyloid beta (Aß) peptide has been linked to the pathology of Alzheimer's disease. A variety of soluble oligomers have been observed to be toxic, ranging from dimers to protofibrils. No tertiary structure has been identified as a single biologically relevant form, though many models are comprised of highly ordered ß-sheets. Evidence exists for much less ordered toxic oligomers. The mechanism of toxicity remains highly debated and probably involves multiple pathways. Interaction of Aß oligomers with the N-terminus of the cellular form of the prion protein (PrP(c)) has recently been proposed. The intrinsically disordered nature of this protein and the highly polymorphic nature of Aß oligomers make structural resolution of the complex exceptionally challenging. In this study, molecular dynamics simulations are performed for dodecameric assemblies of Aß comprised of monomers having a single, short antiparallel ß-hairpin at the C-terminus. The resulting models, devoid of any intermolecular hydrogen bonds, are shown to correlate well with experimental data and are found to be quite stable within the hydrophobic core, whereas the α-helical N-termini transform to a random coil state. This indicates that highly ordered assemblies are not required for stability and less ordered oligomers are a viable component in the population of soluble oligomers. In addition, a tentative model is proposed for the association of Aß dimers with a double deletion mutant of the intrinsically disordered N-terminus of PrP(c). This may be useful as a conceptual working model for the binding of higher order oligomers and in the design of further experiments.


Subject(s)
Amyloid beta-Peptides/chemistry , Models, Molecular , Prions/chemistry , Amyloid beta-Peptides/metabolism , Crystallography, X-Ray , Dimerization , Prions/metabolism , Protein Structure, Tertiary
2.
Comb Chem High Throughput Screen ; 14(4): 267-78, 2011 May.
Article in English | MEDLINE | ID: mdl-21143179

ABSTRACT

Spatially addressable combinatorial libraries were synthesized by solution phase chemistry and screened for binding to human serum albumin. Members of arylidene diamide libraries were among the best hits found, having submicromolar binding affinities. The results were analyzed by the frequency with which particular substituents appeared among the most potent compounds. After immobilization of the ligands either through the oxazolone or the amine substituent, characterization by surface plasmon resonance showed that ibuprofen affected the binding kinetics, but phenylbutazone did not. It is therefore likely that these compounds bind to Site 2 in sub domain IIIA of human serum albumin (HSA).


Subject(s)
Chromatography, Affinity/methods , Combinatorial Chemistry Techniques , Ligands , Serum Albumin/metabolism , Binding Sites/drug effects , Binding, Competitive , Cross-Linking Reagents , High-Throughput Screening Assays , Humans , Ibuprofen/pharmacology , Kinetics , Oxazolone/chemistry , Phenylbutazone/pharmacology , Protein Binding , Protein Structure, Tertiary , Serum Albumin/chemistry , Surface Plasmon Resonance
3.
Nat Chem Biol ; 7(1): 41-50, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21113169

ABSTRACT

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.


Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , B-Lymphocytes/drug effects , Benzamides/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Myeloid Cells/drug effects , Protein Kinase Inhibitors/therapeutic use , Agammaglobulinaemia Tyrosine Kinase , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Autoantibodies/immunology , Autoantibodies/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Benzamides/chemistry , Benzamides/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Interleukin-6/immunology , Interleukin-6/metabolism , Mice , Myeloid Cells/immunology , Myeloid Cells/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/pharmacology , Protein-Tyrosine Kinases/therapeutic use , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
4.
Bioorg Med Chem Lett ; 19(24): 6991-5, 2009 Dec 15.
Article in English | MEDLINE | ID: mdl-19879134

ABSTRACT

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.


Subject(s)
Angiogenesis Inhibitors/chemistry , Imidazoles/chemistry , Phenylurea Compounds/chemistry , Protein Kinase Inhibitors/chemistry , Pyrazines/chemistry , Receptor, EphB4/antagonists & inhibitors , Urea/analogs & derivatives , Angiogenesis Inhibitors/pharmacology , Cell Line, Tumor , Humans , Imidazoles/pharmacology , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology
5.
Curr Opin Drug Discov Devel ; 8(5): 638-45, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16159026

ABSTRACT

Chemical genetics is an important approach in biological research that utilizes small molecules to study protein function. In the context of kinase drug discovery, chemical genetics has broad applications in identifying and validating targets, demonstrating the druggability of a target and providing potential kinase inhibitor leads for further optimization. The successful application of this approach demands that the small-molecule kinase inhibitors used achieve a desired potency and selectivity. However, given the high number (> 518) and homology of kinases in the human genome, identifying potent and selective kinase inhibitors presents a major challenge. This article reviews recent advances in small-molecule kinase inhibitor design, with an emphasis on selectivity, and also discusses recent progress in the development of analog-sensitive kinase allele (ASKA)-based chemical genetics technology, which creates genetically engineered versions of protein kinases that are fully functional and can be selectively inhibited by a unique reference orthogonal inhibitor. Examples of how ASKA technology can be applied to kinase drug discovery is discussed.


Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Phosphotransferases/genetics , Animals , Humans , Molecular Structure , Phosphotransferases/antagonists & inhibitors , Structure-Activity Relationship
6.
Biotechnol Prog ; 21(1): 300-8, 2005.
Article in English | MEDLINE | ID: mdl-15903269

ABSTRACT

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.


Subject(s)
Chromatography, Affinity , Combinatorial Chemistry Techniques/methods , Serum Albumin/chemistry , Combinatorial Chemistry Techniques/economics , Humans , Ligands , Protein Binding/physiology , Sensitivity and Specificity
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