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1.
Nature ; 586(7828): 275-280, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33029001

RESUMO

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.


Assuntos
Organoides/citologia , Organoides/fisiologia , Fenótipo , Regeneração/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/metabolismo , Regeneração/efeitos dos fármacos , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Tretinoína/metabolismo , Vitamina A/farmacologia
2.
Nat Chem Biol ; 11(11): 834-6, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26368590

RESUMO

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Superóxidos/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Animais , Antimicina A/análogos & derivados , Antimicina A/antagonistas & inibidores , Antimicina A/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais , Superóxidos/metabolismo
3.
J Am Chem Soc ; 135(5): 1669-72, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23330637

RESUMO

The identification of factors that promote ß cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent ß cell lines to identify molecules that induce proliferation of ß cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes ß cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of ß cell ablation, WS6 normalized blood glucose and induced concomitant increases in ß cell proliferation and ß cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.


Assuntos
Ensaios de Triagem em Larga Escala , Ilhotas Pancreáticas/efeitos dos fármacos , Ureia/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Estrutura Molecular , Peso Molecular , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/química
4.
J Med Chem ; 63(6): 2958-2973, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32077280

RESUMO

Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).


Assuntos
Compostos Aza/química , Compostos Aza/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Indóis/química , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Compostos Aza/farmacocinética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Hipoglicemiantes/farmacocinética , Indóis/farmacocinética , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Quinases Dyrk
5.
ACS Med Chem Lett ; 8(10): 1048-1053, 2017 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-29057049

RESUMO

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

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