ABSTRACT
N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Drug Design , Pyrrolidinones/pharmacology , Transcription Factors/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , Humans , Models, Molecular , Molecular Structure , Pyrrolidinones/chemistry , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.
Subject(s)
Cell Cycle Proteins/chemistry , Drug Design , Pyrrolidinones/chemical synthesis , Transcription Factors/chemistry , Binding Sites , Cell Cycle Proteins/metabolism , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Gene Expression Regulation/drug effects , Humans , Models, Molecular , Molecular Structure , Protein Binding , Protein Conformation , Pyrrolidinones/chemistry , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Subject(s)
Amino Acids/chemistry , Kisspeptins/agonists , Receptors, G-Protein-Coupled/metabolism , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Ligands , Receptors, G-Protein-Coupled/chemistry , Receptors, Kisspeptin-1ABSTRACT
AIMS: The aim of this study is to evaluate the pathological features, serum hormone levels and ex vivo cultures of pituitary adenomas that occur in rats affected by MENX syndrome. MENX is multiple endocrine neoplasia syndrome caused by a germline mutation in the cell cycle inhibitor p27. Characterization of MENX adenomas is a prerequisite to exploit this animal model for molecular and translational studies of pituitary adenomas. METHODS: We investigated MENX pituitary adenomas with immunohistochemistry, double immunofluorescence, electron microscopy, reverse transcription polymerase chain reaction (RT-PCR), measurement of serum hormone levels and ex vivo cultures. RESULTS: Adenomas in MENX rats belong to the gonadotroph lineage. They start from 4 months of age as multiple neoplastic nodules and progress to become large lesions that efface the gland. Adenomas are composed of chromophobic cells predominantly expressing the glycoprotein alpha-subunit (αGSU). They show mitotic activity and high Ki67 labelling. A few neoplastic cells co-express gonadotropins and the transcription factor steroidogenic factor 1, together with growth hormone or prolactin and Pit-1, suggesting that they are not fully committed to one cell lineage. Ex vivo cultures show features similar to the primary tumour. CONCLUSIONS: Our results suggest that p27 function is critical to regulate gonadotroph cells growth. The MENX syndrome represents a unique model to elucidate the physiological and molecular mechanisms mediating the pathogenesis of gonadotroph adenomas.
Subject(s)
Adenoma/pathology , Cyclin-Dependent Kinase Inhibitor p27/genetics , Multiple Endocrine Neoplasia/pathology , Pituitary Neoplasms/pathology , Adenoma/genetics , Adenoma/metabolism , Animals , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Disease Models, Animal , Fluorescent Antibody Technique , Gonadotropins/genetics , Immunohistochemistry , Multiple Endocrine Neoplasia/genetics , Multiple Endocrine Neoplasia/metabolism , Pituitary Neoplasms/genetics , Pituitary Neoplasms/metabolism , Rats , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A disposable device designed for measuring glycated haemoglobin (hba1c) in human blood was evaluated for use in dogs. edta blood samples were collected from 50 normoglycaemic dogs, 10 dogs suffering from anaemia and 112 diabetic dogs. hba1c was measured in all the dogs except for five of the diabetic animals, in which the concentrations were above the range of the device, that is, more than 13 per cent, and two of the anaemic dogs, in which they were below its limit of detection, that is less than 3 per cent. The diabetic dogs had higher hba1c values (range 4.9 to >13 per cent, median 9.3 per cent) than the normoglycaemic dogs (range 3.7 to 5.6 per cent, median 4.7 per cent). In the anaemic dogs the values were significantly lower (range <3.0 per cent to 5.2 per cent, median 3.5 per cent) than in the normoglycaemic dogs. There was a good correlation (R(2)=0.48) between the measurements obtained with the device and the measurements obtained with a system already validated for use in dogs.
