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1.
Mol Cell Proteomics ; 10(11): M111.009183, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21873567

RESUMEN

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.


Asunto(s)
Ciclopentanos/farmacología , Proteoma/metabolismo , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Cinética , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Proteoma/genética , Proteómica , Interferencia de ARN , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinación
2.
Biochem J ; 430(3): 461-76, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20632995

RESUMEN

The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.


Asunto(s)
Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Secuencia de Aminoácidos , Sitios de Unión , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Células HCT116 , Células HT29 , Humanos , Cinética , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pirazinas/farmacología , Interferencia de ARN , Homología de Secuencia de Aminoácido , Ubiquitina/genética , Ubiquitina/metabolismo
3.
Bioorg Med Chem Lett ; 20(22): 6581-6, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20875739

RESUMEN

Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.


Asunto(s)
Quimotripsina/antagonistas & inhibidores , Dipéptidos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Treonina/química , Humanos , Modelos Moleculares
4.
Bioorg Med Chem Lett ; 20(16): 4800-4, 2010 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-20634068
5.
Sci Rep ; 9(1): 3900, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30846832

RESUMEN

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient's T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo-relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Bioensayo , Linfocitos T/inmunología , Humanos , Activación de Linfocitos/inmunología
6.
Mol Cancer Ther ; 13(6): 1625-35, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24672057

RESUMEN

MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standard-of-care agents and may affect the clinical development of MLN4924. As such, we studied its interaction with other DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in vitro. The combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse xenograft model. Importantly, depletion of genes within the ataxia telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924 results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may provide useful information for the clinical development of these combinations.


Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Ciclopentanos/administración & dosificación , Sinergismo Farmacológico , Mitomicina/administración & dosificación , Pirimidinas/administración & dosificación , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Cromatina/genética , Daño del ADN/efectos de los fármacos , Humanos , Ratones , Enzimas Activadoras de Ubiquitina/genética , Rayos Ultravioleta , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Cancer Res ; 73(1): 225-34, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23100467

RESUMEN

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.


Asunto(s)
Antineoplásicos/farmacología , Ciclopentanos/farmacología , Daño del ADN/efectos de los fármacos , Melanoma/metabolismo , Pirimidinas/farmacología , Ubiquitinas/metabolismo , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Citometría de Flujo , Humanos , Proteína NEDD8 , Reacción en Cadena de la Polimerasa
8.
J Med Chem ; 54(6): 1836-46, 2011 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-21341678

RESUMEN

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.


Asunto(s)
Antineoplásicos/síntesis química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Tetrahidronaftalenos/síntesis química , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Cristalografía por Rayos X , Diseño de Fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/enzimología , Melanoma Experimental/patología , Ratones , Ratones Desnudos , Modelos Moleculares , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Estereoisomerismo , Relación Estructura-Actividad , Tetrahidronaftalenos/química , Tetrahidronaftalenos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Cancer Res ; 70(5): 1970-80, 2010 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-20160034

RESUMEN

The proteasome was validated as an oncology target following the clinical success of VELCADE (bortezomib) for injection for the treatment of multiple myeloma and recurring mantle cell lymphoma. Consequently, several groups are pursuing the development of additional small-molecule proteasome inhibitors for both hematologic and solid tumor indications. Here, we describe MLN9708, a selective, orally bioavailable, second-generation proteasome inhibitor that is in phase I clinical development. MLN9708 has a shorter proteasome dissociation half-life and improved pharmacokinetics, pharmacodynamics, and antitumor activity compared with bortezomib. MLN9708 has a larger blood volume distribution at steady state, and analysis of 20S proteasome inhibition and markers of the unfolded protein response confirmed that MLN9708 has greater pharmacodynamic effects in tissues than bortezomib. MLN9708 showed activity in both solid tumor and hematologic preclinical xenograft models, and we found a correlation between greater pharmacodynamic responses and improved antitumor activity. Moreover, antitumor activity was shown via multiple dosing routes, including oral gavage. Taken together, these data support the clinical development of MLN9708 for both hematologic and solid tumor indications.


Asunto(s)
Compuestos de Boro/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Glicina/análogos & derivados , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteasoma , Animales , Compuestos de Boro/farmacocinética , Ácidos Borónicos/farmacología , Bortezomib , Inhibidores de Cisteína Proteinasa/farmacocinética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Glicina/farmacocinética , Glicina/farmacología , Células HCT116 , Células HT29 , Humanos , Linfoma/tratamiento farmacológico , Linfoma/enzimología , Ratones , Ratones SCID , Complejo de la Endopetidasa Proteasomal/sangre , Pirazinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Cancer Res ; 70(11): 4318-26, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20460535

RESUMEN

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.


Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Muerte Celular/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Pirazinas/farmacología , ARN Interferente Pequeño/genética , Bortezomib , Muerte Celular/genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Daño del ADN , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , Ribosomas/efectos de los fármacos , Serina-Treonina Quinasas TOR , Transfección
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