ABSTRACT
A series of aminomethylpyrazoles were prepared and evaluated using cell-based Smoothened beta-lactamase reporter assay and Smoothened binding assay. Potent Smoothened antagonists 10k and 10l were found to inhibit hair growth in vivo in the C3H/HeN mouse hair growth model. The more selective compound 10l was tested negative in the 3T3 NRU assay, indicating a low risk for causing photo-irritation and was efficacious using the C3H/HeN mouse hair growth model although it was slightly less efficacious than that of the reference compound eflornithine (7).
Subject(s)
Azetidines/chemistry , Hair/drug effects , Pyrazoles/chemistry , Animals , Azetidines/chemical synthesis , Azetidines/pharmacology , Eflornithine/pharmacology , Hair/growth & development , Mice , Mice, Knockout , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Structure-Activity RelationshipABSTRACT
The seven-transmembrane protein Smoothened (Smo) mediates the cellular response to the Hedgehog protein signal and is involved in cell growth and differentiation during embryonic development. Stimulation of the Smo pathway is directly implicated in tissue maintenance and repair, but overactivation of Smo could lead to tumorigenesis. We developed a robust and sensitive functional cell-based assay that measures the activity of endogenous Smo using a beta-lactamase transcriptional readout. This is the first Smo reporter assay that utilizes beta-lactamase reporter technology. This assay type has distinct advantages over other reporter technologies and can be used in a high-throughput mode to search for therapeutically relevant downstream Smo target effectors.
Subject(s)
Receptors, G-Protein-Coupled/genetics , beta-Lactamases/genetics , Animals , Cell Count , Cell Line , Cloning, Molecular , Data Interpretation, Statistical , Dimethyl Sulfoxide/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Enzyme Activation , Flow Cytometry , Genes, Reporter/genetics , Indicators and Reagents , Mice , Plasmids/genetics , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/physiology , Smoothened Receptor , TransfectionABSTRACT
The ligand-gated ion channel family includes receptors for serotonin (5-hydroxytryptamine, 5-HT), acetylcholine, GABA, and glutamate. Drugs targeting subtypes of these receptors have proven useful for the treatment of various neuropsychiatric and neurological disorders. To identify new ligand-gated ion channels as potential therapeutic targets, drafts of human genome sequence were interrogated. Portions of four novel genes homologous to 5-HT(3A) and 5-HT(3B) receptors were identified within human sequence databases. We named the genes 5-HT(3C1)-5-HT(3C4). Radiation hybrid (RH) mapping localized these genes to chromosome 3q27-28. All four genes shared similar intron-exon organizations and predicted protein secondary structure with 5-HT(3A) and 5-HT(3B). Orthologous genes were detected by Southern blotting in several species including dog, cow, and chicken, but not in rodents, suggesting that these novel genes are not present in rodents or are very poorly conserved. Two of the novel genes are predicted to be pseudogenes, but two other genes are transcribed and spliced to form appropriate open reading frames. The 5-HT(3C1) transcript is expressed almost exclusively in small intestine and colon, suggesting a possible role in the serotonin-responsiveness of the gut.
Subject(s)
Chromosomes, Human, Pair 3/genetics , Multigene Family/genetics , Receptors, Serotonin, 5-HT3/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Caco-2 Cells , Cell Line, Tumor , Cloning, Molecular , Exons , Female , Gene Expression , Genes/genetics , Humans , Introns , Male , Molecular Sequence Data , Phylogeny , Radiation Hybrid Mapping , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.