RESUMO
Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos , Timidilato Sintase , Humanos , Células CACO-2 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Timidilato Sintase/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2) , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas de Membrana Transportadoras , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Permeabilidade , Ácido Fólico , Metionina , Carbono/metabolismoRESUMO
A library of chemokine antagonists has been synthesized using a combination of solid and solution-phase chemistry. Structures of known chemokine antagonists were used to produce a pharmacophore which served to guide monomer selection. Several combinations of monomers have resulted in providing novel chemokine antagonists which in some cases display dual chemokine receptor antagonism.
Assuntos
Quimiocinas/antagonistas & inibidores , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas , Animais , Linhagem Celular , Cricetinae , CricetulusRESUMO
Bridgehead metallation is possible in a ketone having the welwistatin skeleton, and this facilitates installation of the isothiocyanate function present in the natural product, and also enables synthesis of remarkable bridgehead alkenes.
Assuntos
Alcaloides/química , Alcenos/química , Modelos Moleculares , Cristalografia por Raios X , Alcaloides Indólicos , Estrutura Molecular , Compostos de Selênio/química , Óxidos de SelênioRESUMO
The currently available therapy for asthma is highly effective and is able to control the disease in the majority of patients. There are two types of treatments for asthma: rapid relief of symptoms, used as needed and long-term control, used on a regular basis. Rapid relief is provided by short-acting beta2-agonists and anticholinergics. The control of asthma is achieved by treatment with inhaled corticosteroids (ICS), theophylline, long acting beta2-agonists and antileukotrienes. Beta2-agonists and corticosteroids dominate asthma therapy, with over 65% of the market share. Corticosteroids are the most effective drugs available to clinicians for the control of inflammation in patients with asthma. ICS have revolutionized the treatment of asthma and are now the first-line treatment for chronic asthma in all ages.
Assuntos
Corticosteroides/uso terapêutico , Antiasmáticos/uso terapêutico , Administração por Inalação , Corticosteroides/administração & dosagem , Corticosteroides/efeitos adversos , Corticosteroides/farmacologia , Antiasmáticos/administração & dosagem , Antiasmáticos/efeitos adversos , Antiasmáticos/farmacologia , Humanos , Patentes como AssuntoRESUMO
[reaction: see text] Catalytic Sc(OTf)(3) greatly increases the efficiency of hydrogen peroxide mediated monooxidation of alkyl-aryl sulfides and methyl cysteine containing peptides. The method is high yielding, compatible with many widely used protecting groups, suitable for solid-phase applications and proceeds with minimum over-oxidation.
RESUMO
[reaction: see text] The partial reduction of 2,5-pyrrole diester 1 followed by enantioselective protonation in situ to furnish synthetically useful building blocks is described. An enantiomeric excess of up to 74% was achieved using (-)-ephedrine and related analogues as chiral proton sources. The pyrroline product obtained could be recrystallized to give enantiomerically pure material.
Assuntos
Técnicas de Química Combinatória , Efedrina/química , Pirróis/química , Catálise , Indicadores e Reagentes , Estrutura Molecular , Oxirredução , Pirróis/análise , Pirróis/síntese química , EstereoisomerismoRESUMO
Dyad beads, bearing both a substrate and a catalyst, were prepared to enable direct split and mix bead based screening for catalysis.
RESUMO
The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca(2+) permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms-the IonWorks(®) Quattro™ and QPatch(®) systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.
Assuntos
Ensaios de Triagem em Larga Escala , Técnicas de Patch-Clamp/métodos , Canais de Cátion TRPC/metabolismo , Animais , Automação , Células CHO , Células Cultivadas , Cricetulus , Humanos , Cinética , Canais de Cátion TRPC/antagonistas & inibidores , Canal de Cátion TRPC6RESUMO
BACKGROUND: Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. METHODS: Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. KEY RESULTS: Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. CONCLUSIONS & INFERENCES: Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.
Assuntos
Hormônios Gastrointestinais/metabolismo , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Transporte de Íons/fisiologia , Neuropeptídeos/metabolismo , Dor Visceral/fisiopatologia , Animais , Cálcio/metabolismo , Colite/metabolismo , Colite/patologia , Feminino , Gânglios Espinais/citologia , Hormônios Gastrointestinais/genética , Trato Gastrointestinal/anatomia & histologia , Humanos , Hiperalgesia/fisiopatologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismoAssuntos
Farmacologia , Receptores de Quimiocinas/efeitos dos fármacos , Animais , Fármacos Anti-HIV/uso terapêutico , Anticorpos Monoclonais/farmacologia , Antagonistas dos Receptores CCR5 , Quimiocinas/farmacologia , Quimiocinas/fisiologia , Desenho de Fármacos , Humanos , Receptores CCR3 , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/química , Receptores de Interleucina-8A/antagonistas & inibidoresRESUMO
A general strategy for the production of pyrrolizidine alkaloids is described, starting from intermediate (+)-9. The key features are diastereoselective dihydroxylation, inversion at the ring junction by hydroboration of an enamine, and ring closure to form the bicyclo ring system. This route is attractive because of its brevity and versatility; four natural products were prepared with differing stereochemistry and substitution patterns. Finally, this work allowed assignment of the absolute stereochemistry of 2,3,7-triepiaustraline and hyacinthacine A 7.
Assuntos
Alcaloides de Pirrolizidina/síntese química , Aminas/química , Hidroxilação , Estrutura Molecular , Alcaloides de Pirrolizidina/química , EstereoisomerismoRESUMO
High throughput screening identified a phenoxyacetic acid scaffold as a novel CRTh2 receptor antagonist chemotype, which could be optimised to furnish a compound with functional potency for inhibition of human eosinophil shape change and oral bioavailability in the rat.
Assuntos
Acetatos/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Acetatos/química , Animais , Disponibilidade Biológica , Eosinófilos/efeitos dos fármacos , Humanos , Ratos , Relação Estrutura-AtividadeRESUMO
Inhibitors of PDE5 are useful therapeutic agents for treatment of erectile dysfunction. A series of novel xanthine derivatives has been identified as potent inhibitors of PDE5, with good levels of selectivity against other PDE isoforms, including PDE6. Studies in the dog indicate excellent oral bioavailability for compound 21.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Xantinas/farmacologia , Animais , Disponibilidade Biológica , Bovinos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Disfunção Erétil/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Masculino , Farmacocinética , Isoformas de Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade , Xantinas/químicaRESUMO
The enzymatic desymmetrization of 2,2- and 2,5-disubstituted pyrroline compounds is reported in a procedure which gives access to both enantiomers in excellent enantiomeric excess and good yield. The enzyme reaction precursors are formed easily from two readily available substituted pyrroles using both ammonia (Na/NH3) and ammonia-free (Li/DBB) Birch reduction conditions.
Assuntos
Lipase/metabolismo , Pirróis/química , Pirróis/metabolismo , Estrutura Molecular , Pseudomonas/enzimologia , EstereoisomerismoRESUMO
A solid-phase approach was used to prepare 20 cystine amide derivatives with disulfide bond formation resulting from an intra-site reaction between neighbouring cysteine residues. Library members were screened as potential organogelators in a range of solvent mixtures and resulted in the identification of a potent gelator able to rigidify water/DMSO mixtures at concentrations as low as 1.3 mM.
Assuntos
Amidas/química , Técnicas de Química Combinatória/métodos , Cistina/análogos & derivados , Cisteína/química , Cistina/química , Géis/química , Estrutura MolecularRESUMO
Ion-extraction mass spectrometry of ladders of mixtures of isotopically labeled compounds from single beads allows the unambiguous sequencing of bead-based peptides and offers significant advantages over traditional methods of library analysis.
Assuntos
Técnicas de Química Combinatória , Biblioteca de Peptídeos , Análise por Conglomerados , Indicadores e Reagentes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PDE5 inhibitors based upon the xanthine scaffold 8 have been expediently synthesized using a solid-phase route. Attachment of the xanthine scaffold 8 using the Rink chloride linker 4 and N-1 functionalization using Mitsunobu chemistry is described. A library of compounds was produced in multi-milligram quantities with excellent purities and acceptable yields. The compounds were tested for their PDE5 inhibitory activity.
Assuntos
Inibidores de Fosfodiesterase/síntese química , Diester Fosfórico Hidrolases/metabolismo , Xantina/química , 3',5'-GMP Cíclico Fosfodiesterases , Técnicas de Química Combinatória , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Bases de Dados Factuais , Indicadores e Reagentes/química , Ressonância Magnética Nuclear Biomolecular , Inibidores de Fosfodiesterase/farmacologia , Relação Estrutura-AtividadeRESUMO
In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.