ABSTRACT
Optimization of lead compound 1, through extensive use of structure-based design and a focus on PI3Kδ potency, isoform selectivity, and inhaled PK properties, led to the discovery of clinical candidates 2 (GSK2269557) and 3 (GSK2292767) for the treatment of respiratory indications via inhalation. Compounds 2 and 3 are both highly selective for PI3Kδ over the closely related isoforms and are active in a disease relevant brown Norway rat acute OVA model of Th2-driven lung inflammation.
Subject(s)
Indazoles/chemistry , Oxazoles/chemistry , Phosphoinositide-3 Kinase Inhibitors , Respiratory Tract Diseases/drug therapy , Sulfonamides/chemistry , Administration, Inhalation , Animals , Asthma/drug therapy , Female , Humans , Indazoles/pharmacokinetics , Indazoles/pharmacology , Indoles , Isoenzymes/antagonists & inhibitors , Male , Microsomes/metabolism , Molecular Docking Simulation , Ovalbumin/immunology , Oxazoles/pharmacokinetics , Oxazoles/pharmacology , Piperazines , Pneumonia/drug therapy , Pneumonia/immunology , Pulmonary Disease, Chronic Obstructive/drug therapy , Rabbits , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Th2 Cells/immunologyABSTRACT
IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.
Subject(s)
Asthma/drug therapy , Cysteine/chemistry , Drug Design , Enzyme Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Animals , Crystallography, X-Ray , Cytokines/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Gene Expression Regulation, Enzymologic , Humans , Jurkat Cells , Leukocytes, Mononuclear/drug effects , Ligands , Male , Particle Size , Protein Binding , Protein-Tyrosine Kinases/chemistry , Rats , Rats, Wistar , Signal TransductionABSTRACT
Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.
Subject(s)
Benzamides/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Mast Cells/drug effects , Pyrazoles/pharmacology , T-Lymphocytes/drug effects , Animals , Cell Line , Cells, Cultured , Cytokines/metabolism , Female , Guinea Pigs , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lung/drug effects , Lung/metabolism , Male , Mast Cells/metabolism , Mice , Mice, Inbred BALB C , Ovalbumin , Rats , Spleen/cytology , T-Lymphocytes/metabolism , Trachea/drug effects , Trachea/physiologyABSTRACT
Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.
