ABSTRACT
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
Subject(s)
Aldehyde Oxidase/metabolism , Janus Kinase Inhibitors/pharmacokinetics , Lung/metabolism , Administration, Intranasal , Administration, Intravenous , Animals , Binding Sites , Drug Delivery Systems , Female , Humans , Janus Kinase Inhibitors/administration & dosage , Janus Kinase Inhibitors/chemical synthesis , Liver/metabolism , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Docking Simulation , Quinazolines/chemical synthesis , Quinazolines/pharmacokinetics , Quinazolines/pharmacology , Structure-Activity RelationshipABSTRACT
ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.
Subject(s)
Aminopeptidases/antagonists & inhibitors , Antigen Presentation/drug effects , Diterpenes, Clerodane/pharmacology , Epitopes/metabolism , Peptides/metabolism , Protease Inhibitors/pharmacology , Allosteric Site , Aminopeptidases/chemistry , Aminopeptidases/metabolism , Animals , Catalytic Domain , Crystallography, X-Ray , Diterpenes, Clerodane/chemistry , Diterpenes, Clerodane/metabolism , Enzyme Activators/chemistry , Enzyme Activators/metabolism , Enzyme Activators/pharmacology , Epitopes/chemistry , HeLa Cells , Humans , Mice , Minor Histocompatibility Antigens/chemistry , Minor Histocompatibility Antigens/metabolism , Peptides/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Binding , Proteolysis/drug effectsABSTRACT
Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBß in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBß alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.
