RESUMO
The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure-activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.
Assuntos
Benzimidazóis/química , Piperazinas/química , Receptores de Glutamato Metabotrópico/metabolismo , Administração Oral , Regulação Alostérica , Animais , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Dopamina/metabolismo , Ensaios de Triagem em Larga Escala , Piperazinas/síntese química , Piperazinas/farmacologia , Ratos , Relação Estrutura-Atividade , Potenciais Sinápticos/efeitos dos fármacosRESUMO
The discovery of drugs for G protein-coupled receptors (GPCRs) has traditionally been very successful, even before the structural nature of these molecular targets was elucidated. Over the years, this family of proteins has become more important in the understanding and treatment of different human pathologies, representing today close to 30% of the molecular targets of all marketed drugs. The sequencing of the human genome unveiled the existence of many new GPCRs and this has increased even more the interest of this family of proteins as potential drug targets. Today the search for compounds that interfere or modulate the function of GPCRs is one of the major focuses of pharmaceutical companies. The understanding of the molecular events that take place upon receptor activation, together with the need of testing large chemical libraries, has resulted in the development of a variety of methods and technologies to measure the activity of these receptors. In this chapter we will review most of the assay technologies currently in use for "in vitro" pharmacological screening, their evolution, their capabilities, and their limitations.
Assuntos
Técnicas de Química Combinatória/métodos , Ensaio Radioligante/métodos , Receptores Acoplados a Proteínas G/fisiologia , Animais , Bioensaio , Avaliação de Medicamentos , HumanosRESUMO
High throughput screening combined with efficient datamining and parallel synthesis led to the discovery of a novel series of indolines showing potent in vitro ghrelin receptor agonist activity and acceleration of gastric emptying in rats.
Assuntos
Indóis/química , Receptores de Grelina/agonistas , Animais , Esvaziamento Gástrico/efeitos dos fármacos , Esvaziamento Gástrico/fisiologia , Hormônio do Crescimento Humano/agonistas , Humanos , Indóis/farmacologia , Ratos , Receptores de Grelina/fisiologia , EstereoisomerismoRESUMO
Multiple sclerosis (MS) is an autoimmune neurodegenerative disease that involves activation of T cells, microglia, and astrocytes. There is a clear unmet medical need for MS, as current therapies reduce the relapse rate, but are unable to prevent the neurological deterioration. Leukemia inhibitory factor (LIF) is a proinflammatory cytokine that can also positively modulate the immune response, by inducing the inhibition of myelin-reactive TH17 differentiation, and by promoting oligodendrocyte-mediated myelination. The aim of this project was to find central nervous system (CNS)-permeable and orally available small molecules that upregulate production of endogenous LIF. We describe here the development of a phenotypic assay and screening of 1.7 million compounds to identify LIF enhancers using U87 MG cells. Five chemically tractable series of compounds and a few singletons were selected for further progression. Some of them were also active in a different LIF-expressing cell line and in primary rat astrocytes. Although further studies would be required to deconvolute the targets involved in LIF induction and to confirm activity of hits in more disease-relevant assays, our results have demonstrated the potential of the phenotypic approach to identify specific and chemically tractable small molecules that trigger the production of LIF in relevant cell lines.
Assuntos
Elementos Facilitadores Genéticos/genética , Fator Inibidor de Leucemia/genética , Esclerose Múltipla/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sistema Nervoso Central/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Celular/efeitos dos fármacos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacosRESUMO
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine associated with multiple diseases, including neurodegenerative disorders. With the ultimate goal of providing novel chemotypes as starting points for development of disease-modifying therapeutics for neurodegeneration, we endeavored to screen the GSK compound collection for MIF inhibitors using a miniaturized, activity-based kinetic assay. The assay monitors the increase in absorbance at 320 nm resulting from keto-to-enol tautomerization of 4-hydroxyphenylpyruvate, a reaction catalyzed by MIF. We ran a full-diversity screen evaluating the inhibitory activity of 1.6 million compounds. Primary hits were confirmed and retested in an orthogonal assay measuring tautomerization of l-dopachrome methyl ester by the decrease in absorbance at 475 nm in kinetic mode. Selected compounds were progressed to medium-throughput mode-of-inhibition studies, which included time dependence, enzyme concentration dependence, and reversibility of their inhibitory effect. With these results and after inspection of the physicochemical properties of compounds, 17 chemotypes were prioritized and progressed to further stages of validation and characterization to better assess their therapeutic potential.
Assuntos
Descoberta de Drogas/métodos , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Doenças Neurodegenerativas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Cinética , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/enzimologia , Doenças Neurodegenerativas/genética , Ácidos Fenilpirúvicos/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.
Assuntos
Isoxazóis/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Cães , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/administração & dosagem , Humanos , Isoxazóis/química , Isoxazóis/farmacocinética , RatosRESUMO
Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.