ABSTRACT
Our first generation CRTH2 and DP dual antagonists, represented by AMG 009, are more potent toward the CRTH2 receptor than to the DP receptor. Here we report our efforts in the discovery of CRTH2 and DP dual antagonists with more balanced potencies to both receptors, such as compound 15.
Subject(s)
Drug Design , Phenylacetates/chemical synthesis , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , HEK293 Cells , Humans , Inhibitory Concentration 50 , Molecular Structure , Phenylacetates/chemistry , Phenylacetates/pharmacology , Protein Binding/drug effectsABSTRACT
We have previously reported that optimization of a series of phenylacetic acid derivatives led to the discovery of CRTH2 and DP dual antagonists, such as AMG 009 and AMG 853. During the optimization process, we discovered that minor structural modifications also afforded potent and selective CRTH2 or DP antagonists. Here we report the structure-activity relationship that led to the discovery of selective CRTH2 antagonists such as 2 and 17, and selective DP antagonists, such as 4 and 5.
Subject(s)
Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Asthma/therapy , Chemistry, Pharmaceutical/methods , Drug Design , Humans , Hypersensitivity/drug therapy , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Phenylacetates/chemistry , Phenylacetates/pharmacology , Prostaglandin D2/metabolism , Receptors, G-Protein-Coupled/metabolism , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
Multiple CRTH2 antagonists are currently evaluated in human clinical trials for asthma and chronic obstructive pulmonary disease (COPD). During our lead optimization for CRTH2 antagonists, an observation of an intramolecular hydrogen bond in ortho-phenylsulfonamido benzophenone derivatives led to the design and synthesis of conformationally constrained benzodiazepinones as potent CRTH2 antagonists. The benzodiazepinones are 2 orders of magnitude more potent than the original flexible bisaryl ethers in our binding assay. Selected benzodiazepinones, such as compound 6, were also potent in the human eosinophil shape change assay. Analysis of the rigid conformations of these benzodiazepinones and ortho-phenylsulfonamido benzophenones provided an explanation for the structure-activity relationship and revealed the possible bound conformations to CRTH2, which may be useful for building a pharmacophore model of CRTH2 antagonists.
ABSTRACT
A series of tetrahydroquinoline-derived inhibitors of the CRTH2 receptor was discovered by a high throughput screen. Optimization of these compounds for potency and pharmacokinetic properties led to the discovery of potent and orally bioavailable CRTH2 antagonists.
Subject(s)
Anti-Allergic Agents/chemistry , Quinolines/chemistry , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Anti-Allergic Agents/pharmacokinetics , Anti-Allergic Agents/pharmacology , Cell Line , Cell Shape/drug effects , Drug Discovery , Eosinophils/drug effects , Eosinophils/immunology , Humans , Quinolines/pharmacokinetics , Quinolines/pharmacologyABSTRACT
The germinal center (GC) is an important site for the generation and selection of B cells bearing high-affinity antibodies, yet GC cell migration and interaction dynamics have not been directly observed. Using two-photon microscopy of mouse lymph nodes, we revealed that GC B cells are highly motile and extend long cell processes. They transited between GC dark and light zones and divided in both regions, although these B cells resided for only several hours in the light zone where antigen is displayed. GC B cells formed few stable contacts with GC T cells despite frequent encounters, and T cells were seen to carry dead B cell blebs. On the basis of these observations, we propose a model in which competition for T cell help plays a more dominant role in the selection of GC B cells than previously appreciated.
Subject(s)
Antibody Affinity , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Germinal Center/immunology , Animals , B-Lymphocytes/physiology , Cell Cycle , Cell Death , Cell Movement , Dendritic Cells, Follicular/cytology , Dendritic Cells, Follicular/physiology , Germinal Center/cytology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Microscopy/methods , Models, Immunological , Mutation , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/physiologyABSTRACT
IgG autoantibodies cause pathology due to their ability to bind self antigens. However, the extent to which the initial B cell activation and isotype switching is antigen-driven is unclear and it has been widely proposed that intrinsic B cell hyperactivity may be a contributing factor. To explore this issue we generated mice with B cell hyperactivity secondary to deficiency in the src kinase Lyn that also expressed a gene-targeted anti-hen egg lysozyme Ig construct (VDJkappa) capable of class switching to all isotypes. The B cell hyperactivity caused spontaneous hypersecretion of antibodies and class switching to IgM, IgA, IgG1 and IgG3 isotypes in the absence of self antigen, and this persisted as an autoimmune phenomenon in the presence of intracellularly expressed hen egg lysozyme. Exaggerated class switching was also unaffected by antigen in vitro. These findings show that systemic high-avidity intracellular self antigens do not induce self tolerance in the face of B cell hyperactivity. Under these circumstances, spontaneous activation of hyperactive B cells leads to isotype switching and the development of high titres of IgG autoantibodies against intracellular proteins.
Subject(s)
Antibody Formation/genetics , Autoimmunity/genetics , B-Lymphocytes/immunology , Immunoglobulin Class Switching , Lymphocyte Activation/genetics , Animals , Autoantigens/immunology , Immunoglobulin Isotypes/genetics , Immunoglobulin Isotypes/metabolism , Mice , Mice, Knockout , Muramidase/immunology , src-Family Kinases/geneticsABSTRACT
The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment.