ABSTRACT
A high-throughput screen against Inventiva's compound library using a Gal4/RORγ-LBD luciferase reporter gene assay led to the discovery of a new series of quinoline sulphonamides as RORγ inhibitors, eventually giving rise to a lead compound having an interesting in vivo profile after oral administration. This lead was evaluated in a target engagement model in mouse, where it reduced IL-17 cytokine production after immune challenge. It also proved to be active in a multiple sclerosis model (EAE) where it reduced the disease score. The synthesis, structure activity relationship (SAR) and biological activity of these derivatives is described herein.
Subject(s)
Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/chemistry , Quinolines/chemistry , Animals , Disease Models, Animal , Humans , MiceABSTRACT
Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.
Subject(s)
Drug Discovery , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Binding Sites/drug effects , Crystallography, X-Ray , High-Throughput Screening Assays , Humans , Mitogen-Activated Protein Kinase 8/chemistry , Mitogen-Activated Protein Kinase 8/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries , Stereoisomerism , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/chemistry , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.
Subject(s)
Anti-Inflammatory Agents/chemistry , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Adenosine Triphosphate/chemistry , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacology , Binding Sites , Binding, Competitive , Computer Simulation , Intracellular Signaling Peptides and Proteins/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.
Subject(s)
Anti-Inflammatory Agents/chemistry , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Administration, Oral , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Binding Sites , Computer Simulation , Crystallography, X-Ray , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolismABSTRACT
We describe the identification, SAR, and in vivo pharmacology of a new series of Src-family selective Lck inhibitors. These thienopyridines were designed based on a desire to access the unique residues in the extended hinge region of Lck.
Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyridines/chemistry , Binding Sites , Drug Design , Humans , Inhibitory Concentration 50 , Pyridines/pharmacology , Structure-Activity Relationship , src-Family Kinases/antagonists & inhibitorsABSTRACT
We describe the identification, SAR, and pharmacology of the src-family selective lck inhibitor A-770041 that prolongs the survival of major histocompatibility mismatched allografts in models of solid organ transplant rejection for greater than 65 days.
Subject(s)
Graft Rejection/prevention & control , Immunosuppressive Agents/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , src-Family Kinases/antagonists & inhibitors , Animals , Chemistry, Pharmaceutical/methods , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacology , Heart Transplantation/methods , Humans , Immunophenotyping , Inhibitory Concentration 50 , Kinetics , Major Histocompatibility Complex , Models, Chemical , Models, Molecular , Pharmaceutical Preparations , Rats , Time Factors , Transplantation, Homologous/methodsABSTRACT
Lck, one of eight members of the Src family of tyrosine kinases, is activated after T cell stimulation and is required for T-cell proliferation and interleukin (IL)-2 production. Inhibition of Lck has been a target to prevent lymphocyte activation and acute rejection. Here, we report the pharmacologic characterization of 1-methyl-1H-indole-2-carboxylic acid (4-{1-[4-(4-acetyl-piperazin-l-yl)-cyclohexyl]-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl}-2-methoxy-phenyl)-amide (A-770041), an orally bioavailable pyrazolo[3,4-d]pyrimidine with increased selectivity for Lck compared with previously reported compounds. A-770041 is a 147 nM inhibitor of Lck (1 mM ATP) and is 300-fold selective against Fyn, the other Src family kinase involved in T-cell signaling. Concanavalin A-stimulated IL-2 production in whole blood is inhibited by A-770041 with an EC50 of approximately 80 nM. A-770041 is orally bioavailable (F = 34.1 +/- 7.2% at 10 mg/kg) and has a t(1/2) of 4.1 +/- 0.1 h. Concanavalin A-induced IL-2 production in vivo is inhibited by oral administration of A-770041 (in vivo EC50 = 78 +/- 28 nM). Doses of A-770041 at or above 10 mg/kg/day prevent rejection of hearts transplanted heterotopically in rats from Brown Norway donors to Lewis recipients across a major histocompatibility barrier for least 65 days. Grafts from animals treated with 20 mg/kg/day A-770041 or 10 mg/day Cyclosporin A had minimal microvascular changes or multifocal mononuclear infiltrates. However, mineralization in myocytes from the grafts from A-770041-treated animals was less than animals treated with Cyclosporin A. Lck inhibition is an attractive target to prevent acute rejection.
Subject(s)
Graft Rejection/prevention & control , Heart Transplantation/adverse effects , Intracellular Signaling Peptides and Proteins/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Animals , Interleukin-2/metabolism , Intracellular Signaling Peptides and Proteins/pharmacokinetics , Lymphocyte Activation/drug effects , Male , Rats , Rats, Inbred BN , Rats, Inbred Lew , T-Lymphocytes/immunology , Transplantation, HomologousABSTRACT
We have identified the pyrazolo[3,4-d]pyrimidine A-420983 (compound 7) as a potent inhibitor of lck. A-420983 exhibits oral efficacy in animal models of delayed-type hypersensitivity and organ transplant rejection.
Subject(s)
Graft Rejection/drug therapy , Immunosuppressive Agents/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Administration, Oral , Animals , Dogs , Heart Transplantation/adverse effects , Humans , Hypersensitivity, Delayed/drug therapy , Immunosuppressive Agents/pharmacokinetics , Immunosuppressive Agents/therapeutic use , Inhibitory Concentration 50 , Interleukin-2/biosynthesis , Mice , Mice, Inbred C57BL , Microsomes, Liver , Models, Animal , Models, Molecular , Molecular Structure , Pyrazoles/administration & dosage , Pyrazoles/pharmacokinetics , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Structure-Activity RelationshipABSTRACT
A series of pyrrolo[2,3-d]pyrimidines was synthesized and evaluated as inhibitors of Lck. Lck accommodates a diverse set of substituents at N-7. Altering the substituent at N-7 provided compound 13, an orally available lck inhibitor which inhibited TCR mediated IL-2 production after oral dosing.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/analysis , Pyrimidines/chemistry , Pyrimidines/pharmacology , Humans , Jurkat CellsABSTRACT
A series of para-substituted 3-phenyl pyrazolopyrimidines was synthesized and evaluated as inhibitors of lck. The nature of the substitution affected enzyme selectivity and potency for lck, src, kdr, and tie-2. The para-phenoxyphenyl analogue 2 is an orally active lck inhibitor with a bioavailability of 69% and exhibits an extended duration of action in animal models of T cell inhibition.