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1.
Elife ; 122023 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-36867026

RESUMEN

Imaging endogenous mRNAs in live animals is technically challenging. Here, we describe an MS2-based signal amplification with the Suntag system that enables live-cell RNA imaging of high temporal resolution and with 8xMS2 stem-loops, which overcomes the obstacle of inserting a 1300 nt 24xMS2 into the genome for the imaging of endogenous mRNAs. Using this tool, we were able to image the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans.


Asunto(s)
Caenorhabditis elegans , ARN , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Diagnóstico por Imagen , Expresión Génica
2.
J Am Chem Soc ; 143(37): 15073-15083, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34520194

RESUMEN

Proteolysis targeting chimeras (PROTACs) represent a new class of promising therapeutic modalities. PROTACs hijack E3 ligases and the ubiquitin-proteasome system (UPS), leading to selective degradation of the target proteins. However, only a very limited number of E3 ligases have been leveraged to generate effective PROTACs. Herein, we report that the KEAP1 E3 ligase can be harnessed for targeted protein degradation utilizing a highly selective, noncovalent small-molecule KEAP1 binder. We generated a proof-of-concept PROTAC, MS83, by linking the KEAP1 ligand to a BRD4/3/2 binder. MS83 effectively reduces protein levels of BRD4 and BRD3, but not BRD2, in cells in a concentration-, time-, KEAP1- and UPS-dependent manner. Interestingly, MS83 degrades BRD4/3 more durably than the CRBN-recruiting PROTAC dBET1 in MDA-MB-468 cells and selectively degrades BRD4 short isoform over long isoform in MDA-MB-231 cells. It also displays improved antiproliferative activity than dBET1. Overall, our study expands the limited toolbox for targeted protein degradation.


Asunto(s)
Antineoplásicos , Proteína 1 Asociada A ECH Tipo Kelch , Humanos , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Modelos Moleculares , Proteolisis , Neoplasias de la Mama Triple Negativas
3.
J Med Chem ; 63(24): 15883-15905, 2020 12 24.
Artículo en Inglés | MEDLINE | ID: mdl-33284613

RESUMEN

Previously, we reported a first-in-class von Hippel-Lindau (VHL)-recruiting mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) degrader, MS432. To date, only two MEK1/2 degrader papers have been published and very limited structure-activity relationships (SAR) have been reported. Here, we describe our extensive SAR studies exploring both von Hippel-Lindau (VHL) and cereblon (CRBN) E3 ligase ligands and a variety of linkers, which resulted in two novel, improved VHL-recruiting MEK1/2 degraders, 24 (MS928) and 27 (MS934), and the first CRBN-recruiting MEK1/2 degrader 50 (MS910). These compounds potently and selectively degraded MEK1/2 by hijacking the ubiquitin-proteasome system, inhibited downstream signaling, and suppressed cancer cell proliferation. Furthermore, concurrent inhibition of BRAF or PI3K significantly potentiated the antitumor activity of degrader 27, suggesting that the combination of MEK1/2 degradation with BRAF or PI3K inhibition may provide potential therapeutic benefits. Finally, besides being more potent, degrader 27 displayed improved plasma exposure levels in mice, representing the best MEK1/2 degrader to date for in vivo studies.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Proliferación Celular , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteolisis , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirrolidinas/química , Pirrolidinas/farmacocinética , Transducción de Señal , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacocinética , Distribución Tisular , Células Tumorales Cultivadas , Ubiquitina/metabolismo
4.
Nat Chem Biol ; 16(2): 214-222, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31819273

RESUMEN

The enhancer of zeste homolog 2 (EZH2) is the main enzymatic subunit of the PRC2 complex, which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to promote transcriptional silencing. EZH2 is overexpressed in multiple types of cancer including triple-negative breast cancer (TNBC), and high expression levels correlate with poor prognosis. Several EZH2 inhibitors, which inhibit the methyltransferase activity of EZH2, have shown promise in treating sarcoma and follicular lymphoma in clinics. However, EZH2 inhibitors are ineffective at blocking proliferation of TNBC cells, even though they effectively reduce the H3K27me3 mark. Using a hydrophobic tagging approach, we generated MS1943, a first-in-class EZH2 selective degrader that effectively reduces EZH2 levels in cells. Importantly, MS1943 has a profound cytotoxic effect in multiple TNBC cells, while sparing normal cells, and is efficacious in vivo, suggesting that pharmacologic degradation of EZH2 can be advantageous for treating the cancers that are dependent on EZH2.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Piperazinas/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/farmacocinética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Técnicas de Inactivación de Genes , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos BALB C , Terapia Molecular Dirigida , Proteolisis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
5.
J Med Chem ; 62(23): 10897-10911, 2019 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-31730343

RESUMEN

MEK1 and MEK2 (also known as MAP2K1 and MAP2K2) are the "gatekeepers" of the ERK signaling output with redundant roles in controlling ERK activity. Numerous inhibitors targeting MEK1/2 have been developed including three FDA-approved drugs. However, acquired resistance to MEK1/2 inhibitors has been observed in patients, and new therapeutic strategies are needed to overcome the resistance. Here, we report a first-in-class degrader of MEK1/2, MS432 (23), which potently and selectively degraded MEK1 and MEK2 in a VHL E3 ligase- and proteasome-dependent manner and suppressed ERK phosphorylation in cells. It inhibited colorectal cancer and melanoma cell proliferation much more effectively than its negative control MS432N (24), and its effect was phenocopied by MEK1/2 knockdown. Compound 23 was highly selective for MEK1/2 in global proteomic profiling studies. It was also bioavailable in mice and can be used for in vivo efficacy studies. We provide two well-characterized chemical tools to the biomedical community.


Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Inhibidores Enzimáticos/química , Células HT29 , Humanos , Estructura Molecular
6.
FEBS Lett ; 591(16): 2379-2393, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28726242

RESUMEN

Microtubules (MTs) play essential roles in many steps of autophagy, an important degradation pathway in the maintenance of cellular homoeostasis. In many cells, MT networks are comprised of centrosomal MTs and noncentrosomal MTs. However, it is unknown whether noncentrosomal MTs and its binding proteins are involved in autophagy. Here, we show in HeLa cells that calmodulin-regulated spectrin-associated protein 2 (CAMSAP2), a noncentrosomal MT minus-end stabilizing protein, regulates retrograde transport of autophagosomes through MT dynamics. CAMSAP2 cooperates with EB1 to regulate end-binding protein 1 (EB1) behaviour at MT plus ends, MT growth directions and autophagosome transport. An association between CAMSAP2 and EB1 in the cytosol may modulate EB1 binding to MT plus ends. Collectively, our data indicate that noncentrosomal MTs regulate autophagy through a cross-talk between CAMSAP2 and EB1.


Asunto(s)
Autofagosomas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Autofagia , Transporte Biológico , Citosol/metabolismo , Células HeLa , Humanos , Unión Proteica
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