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1.
J Biol Chem ; 287(19): 15512-22, 2012 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-22427669

RESUMEN

Uba6 is a homolog of the ubiquitin-activating enzyme, Uba1, and activates two ubiquitin-like proteins (UBLs), ubiquitin and FAT10. In this study, biochemical and biophysical experiments were performed to understand the mechanisms of how Uba6 recognizes two distinct UBLs and catalyzes their activation and transfer. Uba6 is shown to undergo a three-step activation process and form a ternary complex with both UBLs, similar to what has been observed for Uba1. The catalytic mechanism of Uba6 is further supported by inhibition studies using a mechanism-based E1 inhibitor, Compound 1, which forms covalent adducts with both ubiquitin and FAT10. In addition, pre-steady state kinetic analysis revealed that the rates of UBL-adenylate (step 1) and thioester (step 2) formation are similar between ubiquitin and FAT10. However, distinct kinetic behaviors were also observed for ubiquitin and FAT10. FAT10 binds Uba6 with much higher affinity than ubiquitin while demonstrating lower catalytic activity in both ATP-PP(i) exchange and E1-E2 transthiolation assays. Also, Compound 1 is less potent with FAT10 as the UBL compared with ubiquitin in ATP-PP(i) exchange assays, and both a slow rate of covalent adduct formation and weak adduct binding to Uba6 contribute to the diminished potency observed for FAT10. Together with expression level analysis in IM-9 cells, this study sheds light on the potential role of cytokine-induced FAT10 expression in regulating Uba6 pathways.


Asunto(s)
Adenosina Trifosfato/metabolismo , Difosfatos/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Activación Enzimática/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica/efectos de los fármacos , Spodoptera , Especificidad por Sustrato , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Resonancia por Plasmón de Superficie , Factor de Necrosis Tumoral alfa/farmacología , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Ubiquitinas/química , Ubiquitinas/genética
2.
Nat Med ; 24(2): 186-193, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29334375

RESUMEN

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.


Asunto(s)
Neoplasias/tratamiento farmacológico , Nucleósidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Humanos , Imidas/farmacología , Ratones , Neoplasias/genética , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Pirazoles , Pirimidinas , Sulfuros , Ubiquitina/antagonistas & inhibidores , Ubiquitina/química , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Cancer Cell ; 21(3): 388-401, 2012 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-22439935

RESUMEN

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEß as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEß mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEß mutations.


Asunto(s)
Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Mutación , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/genética , Animales , Sitios de Unión , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ratones , Ratones Desnudos , Ratas , Ratas Desnudas , Células Tumorales Cultivadas , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Mol Cancer Ther ; 11(9): 2045-53, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22609854

RESUMEN

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Antineoplásicos/administración & dosificación , Benzazepinas/administración & dosificación , Proteínas de Ciclo Celular/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tionas/administración & dosificación , Administración Oral , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Benzazepinas/farmacocinética , Benzazepinas/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Rituximab , Tionas/farmacocinética , Tionas/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1
5.
Cancer Res ; 71(8): 3042-51, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21487042

RESUMEN

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.


Asunto(s)
Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Ubiquitinas/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Daño del ADN , ADN de Neoplasias/biosíntesis , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Proteína NEDD8 , Pirimidinas/farmacología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Fase S , Ubiquitinas/genética , Ubiquitinas/metabolismo
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