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1.
iScience ; 27(5): 109693, 2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38689642

RESUMEN

The USP7 deubiquitinase regulates proteins involved in the cell cycle, DNA repair, and epigenetics and has been implicated in cancer progression. USP7 inhibition has been pursued for the development of anti-cancer therapies. Here, we describe the discovery of potent and specific USP7 inhibitors exemplified by FX1-5303. FX1-5303 was used as a chemical probe to study the USP7-mediated regulation of p53 signaling in cells. It demonstrates mechanistic differences compared to MDM2 antagonists, a related class of anti-tumor agents that act along the same pathway. FX1-5303 synergizes with the clinically approved BCL2 inhibitor venetoclax in acute myeloid leukemia (AML) cell lines and ex vivo patient samples and leads to strong tumor growth inhibition in in vivo mouse xenograft models of multiple myeloma and AML. This work introduces new USP7 inhibitors, differentiates their mechanism of action from MDM2 inhibition, and identifies specific opportunities for their use in the treatment of AML.

2.
Nat Genet ; 49(3): 465-469, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28166211

RESUMEN

Germline mutations in Ras pathway components are associated with a large class of human developmental abnormalities, known as RASopathies, that are characterized by a range of structural and functional phenotypes, including cardiac defects and neurocognitive delays. Although it is generally believed that RASopathies are caused by altered levels of pathway activation, the signaling changes in developing tissues remain largely unknown. We used assays with spatiotemporal resolution in Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish) to quantify signaling changes caused by mutations in MAP2K1 (encoding MEK), a core component of the Ras pathway that is mutated in both RASopathies and cancers in humans. Surprisingly, we discovered that intrinsically active MEK variants can both increase and reduce the levels of pathway activation in vivo. The sign of the effect depends on cellular context, implying that some of the emerging phenotypes in RASopathies may be caused by increased, as well as attenuated, levels of Ras signaling.


Asunto(s)
Mutación de Línea Germinal/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Transducción de Señal/genética , Proteínas ras/genética , Animales , Drosophila melanogaster/genética , Cardiopatías/genética , Humanos , Neoplasias/genética , Trastornos Neurocognitivos/genética , Fenotipo , Pez Cebra/genética
3.
Proc Natl Acad Sci U S A ; 114(3): 510-515, 2017 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-28049852

RESUMEN

Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.


Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas ras/genética , Animales , Animales Modificados Genéticamente , Discapacidades del Desarrollo/tratamiento farmacológico , Discapacidades del Desarrollo/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Mutación , Fenotipo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(28): 8590-5, 2015 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-26124095

RESUMEN

Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.


Asunto(s)
Proteínas de Drosophila/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HMGB/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Drosophila , Proteínas de Drosophila/química , Proteínas HMGB/química , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Procesos Fotoquímicos , Proteínas Represoras/química
5.
Curr Biol ; 23(21): R972-9, 2013 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-24200329

RESUMEN

A key step towards a chemical picture of enzyme catalysis was taken in 1913, when Leonor Michaelis and Maud Menten published their studies of sucrose hydrolysis by invertase. Based on a novel experimental design and a mathematical model, their work offered a quantitative view of biochemical kinetics well before the protein nature of enzymes was established and complexes with substrates could be detected. Michaelis-Menten kinetics provides a solid framework for enzyme kinetics in vitro, but what about kinetics in cells, where enzymes can be highly regulated and participate in a multitude of interactions? We discuss this question using the Extracellular Signal Regulated Kinase (ERK), which controls a myriad functions in cells, as a model of an important enzyme for which we have crystal structures, quantitative in vitro assays, and a vast list of binding partners. Despite great progress, we still cannot quantitatively predict how the rates of ERK-dependent reactions respond to genetic and pharmacological perturbations. Achieving this goal, which is important from both fundamental and practical standpoints, requires measuring the rates of enzyme reactions in their native environment and interpreting these measurements using simple but realistic mathematical models--the two elements which served as the cornerstones for Michaelis' and Menten's seminal 1913 paper.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/química , Modelos Biológicos , Modelos Químicos , Animales , Catálisis , Humanos , Cinética , Biología de Sistemas
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