ABSTRACT
Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/metabolism , Small Molecule Libraries/pharmacology , Carrier Proteins , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins/chemistry , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/chemistry , Protein Binding/drug effects , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , ThermodynamicsABSTRACT
TWEAK (TNF homologue with weak apoptosis-inducing activity) and Fn14 (fibroblast growth factor-inducible protein 14) are members of the tumor necrosis factor (TNF) ligand and receptor super-families. Having observed that Xenopus Fn14 cross-reacts with human TWEAK, despite its relatively low sequence homology to human Fn14, we examined the conservation in tertiary fold and binding interfaces between the two species. Our results, combining NMR solution structure determination, binding assays, extensive site-directed mutagenesis and molecular modeling, reveal that, in addition to the known and previously characterized ß-hairpin motif, the helix-loop-helix motif makes an essential contribution to the receptor/ligand binding interface. We further discuss the insight provided by the structural analyses regarding how the cysteine-rich domains of the TNF receptor super-family may have evolved over time. DATABASE: Structural data are available in the Protein Data Bank/BioMagResBank databases under the accession codes 2KMZ, 2KN0 and 2KN1 and 17237, 17247 and 17252. STRUCTURED DIGITAL ABSTRACT: TWEAK binds to hFn14 by surface plasmon resonance (View interaction) xeFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction) TWEAK binds to xeFn14 by surface plasmon resonance (View interaction) hFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction).
Subject(s)
Receptors, Tumor Necrosis Factor/chemistry , Tumor Necrosis Factors/chemistry , Xenopus Proteins/chemistry , Amino Acid Sequence , Animals , Cytokine TWEAK , Extracellular Space/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , TWEAK Receptor , Xenopus laevisABSTRACT
The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.
Subject(s)
I-kappa B Kinase/chemistry , Amino Acid Sequence , Binding Sites , Biophysics/methods , Dimerization , Escherichia coli/genetics , Humans , Hydrophobic and Hydrophilic Interactions , I-kappa B Kinase/isolation & purification , I-kappa B Kinase/metabolism , Inclusion Bodies/chemistry , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrum Analysis, RamanABSTRACT
Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.
Subject(s)
Antibodies, Blocking/pharmacology , Cytokines/pharmacology , Immunoglobulin Fab Fragments/pharmacology , Monocyte Chemoattractant Proteins/pharmacology , Receptors, Chemokine/drug effects , Amino Acid Sequence , Animals , Antibodies, Blocking/immunology , Binding Sites , Chemokine CCL2/chemistry , Chemokine CCL2/pharmacology , Chemokine CCL7 , Chemokine CCL8 , Cytokines/metabolism , Humans , Immunoglobulin Fab Fragments/immunology , Inflammation/drug therapy , Mice , Models, Molecular , Molecular Sequence Data , Monocyte Chemoattractant Proteins/chemistry , Mutation , Receptors, CCR2 , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/chemistry , Structure-Activity Relationship , Wounds and Injuries/drug therapyABSTRACT
Asthma, a chronic inflammatory disease of the airways, is a significant burden on our healthcare system. There is high unmet need for treatments directed towards the underlying causes of the disease. The cell surface integrin VLA-4 (very late antigen-4; alpha4beta1; CD49d/CD29) plays an important role in the trafficking of white blood cells to sites of inflammation and represents an exciting target for the development of novel anti-inflammatory drugs for the treatment of asthma. Here, we review our efforts to use rational design to identify potent, selective inhibitors of VLA-4. We describe the discovery of a series of potent VLA-4 inhibitors through the addition of a novel N-terminal organic cap to a tetrapeptide VLA-4 binding motif 4-((N'-2-methylphenyl)uriedo)phenylacetyl-Leu-Asp-Val-Pro ; Kd = 70 pM), and rationalize their structure-activity relationships using 3D-QSAR. Also, we show our rational peptidomimetic design strategy using "template hopping" from the gpIIb/IIIa integrin antagonist field, and also a novel virtual screening strategy. Two series have been developed, one that has high selectivity for the activated over the non-activated state of the receptor, and the other which is non-selective inhibiting both activated and non-activated VLA-4. Both series are highly selective for VLA-4 versus against other integrin family members. These inhibitors show promise in the treatment of asthma, based upon efficacy in a sheep model of asthma, where they inhibit both the early and late-phase responses to asthma and also block hypersensitivity.
Subject(s)
Asthma/drug therapy , Integrin alpha4beta1/antagonists & inhibitors , Amides/chemistry , Amides/pharmacology , Amides/therapeutic use , Animals , Drug Design , Humans , Peptides/chemistry , Peptides/pharmacology , Peptides/therapeutic use , Structure-Activity RelationshipABSTRACT
La valoración preoperatoria cardiovascular es importante para la identificación de pacientes con riesgo de isquemia perioperatoria. Desde hace años se han descrito diferentes índices de riesgo que han servido de base para la elaboración de diagramas de flujo más completos como el diseñado recientemente por el COLEGIO AMERICANO DE CARDIOLOGOS Y LA ASOCIACION AMERICANA DEL CORAZON (ACC & AHA) y que se presenta a continuación. Además, se resumen los principales exámenes paraclínicos que se deben utilizar para la detección de pacientes con enfermedad coronaria y que deben ser llevados a cirugía no cardíaca
