ABSTRACT
Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, â¼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.
Subject(s)
Phosphotransferases/isolation & purification , Protein Kinase Inhibitors/pharmacology , Proteomics , Pyrimidines/chemistry , ortho-Aminobenzoates/chemistry , Cell Line , Chromatography, Liquid/methods , Humans , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Tandem Mass Spectrometry/methodsABSTRACT
The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.
Subject(s)
Pyrimidines/chemistry , Receptors, Neuropeptide Y/antagonists & inhibitors , Spiro Compounds/chemistry , Amides/chemical synthesis , Amides/chemistry , Amides/pharmacokinetics , Amines/chemical synthesis , Amines/chemistry , Amines/pharmacokinetics , Animals , Central Nervous System/drug effects , Humans , Microsomes, Liver/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Rats , Receptors, Neuropeptide Y/metabolism , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The identification of a highly selective D(2) partial agonist, D(3) antagonist tool molecule which demonstrates high levels of brain exposure and selectivity against an extensive range of dopamine, serotonin, adrenergic, histamine, and muscarinic receptors is described.
Subject(s)
Brain/drug effects , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , Receptors, Dopamine D2/agonists , Receptors, Dopamine D3/antagonists & inhibitors , Animals , Brain/metabolismABSTRACT
A new class of selective MMP-12 inhibitors have been identified via high throughput screening. Crystallization with MMP-12 confirmed the mode of binding and allowed initial optimization to be carried out using classical structure based design.
Subject(s)
Carboxylic Acids/chemistry , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemistry , Animals , Binding Sites , Carboxylic Acids/chemical synthesis , Carboxylic Acids/pharmacology , Crystallography, X-Ray , Drug Design , Guinea Pigs , Humans , Matrix Metalloproteinase 12/metabolism , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
A novel small molecule NPY Y2 antagonist (3) identified from high throughput screening is described. A subsequent SAR study and optimisation programme based around this molecule is also described, leading to the identification of potent and soluble pyridyl analogue 36.
Subject(s)
Amides/chemistry , Arginine/analogs & derivatives , Benzazepines/pharmacology , Receptors, Neuropeptide Y/antagonists & inhibitors , Receptors, Neuropeptide Y/chemistry , Animals , Arginine/pharmacology , Chemistry, Organic/methods , Chemistry, Pharmaceutical/methods , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Peptide Fragments/chemistry , Protein Binding , Rats , Structure-Activity RelationshipABSTRACT
The synthesis and SAR of a new series of potent and selective dopamine D(3) receptor antagonists is reported.
Subject(s)
Imidazolidines/chemistry , Positron-Emission Tomography , Receptors, Dopamine D3/antagonists & inhibitors , Animals , Cats , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Imidazolidines/chemical synthesis , Imidazolidines/pharmacology , Ligands , Receptors, Dopamine D3/metabolism , Structure-Activity RelationshipSubject(s)
Indoles/chemistry , Palladium/chemistry , Carbon/chemistry , Catalysis , Nitrogen/chemistryABSTRACT
Over the past two decades, significant advances have been made towards developing chemically catalyzed asymmetric cyanohydrin syntheses. Preparations that were classically highly substrate specific, often using stoichiometric quantities of reagents, have been revolutionized by a new generation of catalysts. Methods currently available rival, and in many cases surpass, enzymatic procedures in terms of synthetic utility, generic applicability, and enantioselectivity. Such protocols are increasingly finding application in the syntheses of both biologically active natural products and therapeutically important synthetic compounds.
