ABSTRACT
Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60-70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Pyrazoles/pharmacology , Pyridines/pharmacology , Pyrrolidines/pharmacology , Aminophenols/pharmacology , Aminopyridines/pharmacology , Benzodioxoles/pharmacology , Bronchi/drug effects , Bronchi/metabolism , Chloride Channels/genetics , Chloride Channels/metabolism , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Drug Combinations , Epithelial Cells/metabolism , Humans , Indoles/pharmacology , Protein Folding/drug effects , Pyrazoles/metabolism , Pyridines/metabolism , Pyrrolidines/metabolism , Quinolines/pharmacologyABSTRACT
Due to the complex and multifactorial nature of bipolar disorder (BD), single-target drugs have traditionally provided limited relief with no disease-modifying effects. In line with the polypharmacology paradigm, we attempted to overcome these limitations by devising two series of multitarget-directed ligands endowed with both a partial agonist profile at dopamine receptor D3 (D3R) and inhibitory activity against glycogen synthase kinase 3 beta (GSK-3ß). These are two structurally unrelated targets that play independent, yet connected, roles in cognition and mood regulation. Two compounds (7 and 10) emerged as promising D3R/GSK-3ß multitarget-directed ligands with nanomolar activity at D3R and low-micromolar inhibition of GSK-3ß, thereby confirming, albeit preliminarily, the feasibility of our strategy. Furthermore, 7 showed promising drug-like properties in stability and pharmacokinetic studies.
Subject(s)
Antipsychotic Agents/pharmacokinetics , Bipolar Disorder/drug therapy , Drug Design , Antipsychotic Agents/chemical synthesis , Antipsychotic Agents/chemistry , Bipolar Disorder/metabolism , Dose-Response Relationship, Drug , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Molecular Structure , Receptors, Dopamine D3/agonists , Receptors, Dopamine D3/metabolism , Structure-Activity RelationshipABSTRACT
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability.
Subject(s)
Benzamidines/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/drug therapy , DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Phenylalanine/metabolism , Thiadiazoles/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , DNA-Binding Proteins/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Epithelial Cells/metabolism , Humans , Mice , Models, Molecular , Molecular Structure , Phenylalanine/genetics , Structure-Activity Relationship , Ubiquitin-Protein Ligases/metabolismABSTRACT
Proteinase activated receptor-2 plays a crucial role in a wide variety of conditions with a strong inflammatory component. We present the discovery and characterization of two structurally different, potent, selective, and metabolically stable small-molecule PAR-2 agonists. These ligands may be useful as pharmacological tools for elucidating the complex physiological role of the PAR-2 receptors as well as for the development of PAR-2 antagonists.
