ABSTRACT
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Subject(s)
Drug Design , Drug Inverse Agonism , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pyridines/administration & dosage , Pyridines/pharmacology , Administration, Oral , Animals , Biological Availability , Drug Evaluation, Preclinical , Humans , Mice , Pyridines/pharmacokinetics , Th17 Cells/drug effects , Th17 Cells/metabolismABSTRACT
A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.
Subject(s)
Chemistry Techniques, Analytical/methods , Environmental Monitoring/methods , Estrogen Receptor alpha/chemistry , Estrogens/analysis , Water Pollutants, Chemical/analysis , Estrogen Receptor alpha/isolation & purification , Fluorescent Dyes/chemistry , Humans , Ligands , Protein Binding , Staining and LabelingABSTRACT
A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRalpha than on LXRbeta. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.
Subject(s)
Arteriosclerosis/drug therapy , DNA-Binding Proteins/agonists , Indazoles/pharmacology , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Triglycerides/biosynthesis , Animals , Arteriosclerosis/metabolism , Cell Differentiation/drug effects , Cell Line , Cricetinae , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Humans , Hydrogen Bonding , Indazoles/chemical synthesis , Indazoles/chemistry , Ligands , Liver/drug effects , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Models, Molecular , Molecular Structure , Orphan Nuclear Receptors , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Structure-Activity Relationship , Triglycerides/bloodABSTRACT
A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.
Subject(s)
Anticholesteremic Agents/chemical synthesis , Atherosclerosis/drug therapy , DNA-Binding Proteins/agonists , Phenylacetates/chemical synthesis , Quinolines/chemical synthesis , Receptors, Cytoplasmic and Nuclear/agonists , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/biosynthesis , Animals , Anticholesteremic Agents/chemistry , Anticholesteremic Agents/pharmacology , Binding Sites , Biological Availability , Cell Line , Cholesterol/metabolism , DNA-Binding Proteins/genetics , Drug Stability , Female , Humans , In Vitro Techniques , Ligands , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Orphan Nuclear Receptors , Phenylacetates/chemistry , Phenylacetates/pharmacology , Protein Structure, Tertiary , Quinolines/chemistry , Quinolines/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Structure-Activity Relationship , Transcriptional ActivationABSTRACT
The structures of the liver X receptor LXRbeta (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 A, respectively. Together with its isoform LXRalpha (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRbeta structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed "agonist conformation" of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor.
