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1.
J Med Chem ; 61(22): 10053-10066, 2018 11 21.
Artículo en Inglés | MEDLINE | ID: mdl-30373366

RESUMEN

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.


Asunto(s)
Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Diseño de Fármacos , Plasmodium falciparum/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos/efectos de los fármacos , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química
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.
J Biol Chem ; 289(33): 22648-22658, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-24966333

RESUMEN

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.


Asunto(s)
Complejos Multiproteicos , Proteínas , Enzimas Activadoras de Ubiquitina , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Línea Celular , Activación Enzimática , Humanos , Modelos Químicos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo
4.
Methods Mol Biol ; 832: 577-88, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22350913

RESUMEN

The NEDD8 conjugation pathway is initiated by the NEDD8 E1, also known as NEDD8 activating enzyme (NAE) or APPBP1/UBA3 (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). The best described biological role for NEDD8 conjugation is to regulate the activity of the cullin RING ligase (CRL) family of ubiquitin E3 ligases (Gong, Yeh. J Biol Chem 274:12063-12042, 1999). In this way, the NEDD8 pathway regulates the turnover of a subset of ubiquitin proteasome system (UPS) substrates that are essential for cancer cell growth and survival (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). We recently initiated clinical trials with a first-in-class small molecule inhibitor of NAE for the treatment of cancer (Soucy, Smith, Milhollen. Nature 458:732-737, 2009). Here we describe a biochemical and cell-based assay used to identify NAE inhibitors and monitor inhibition of the NEDD8 conjugation pathway.


Asunto(s)
Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Ubiquitinas/metabolismo , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Activación Enzimática , Humanos , Proteína NEDD8 , Ubiquitinas/análisis
5.
J Biol Chem ; 286(47): 40867-77, 2011 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-21969368

RESUMEN

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2). MLN4924 is an adenosine sulfamate analogue that was identified as a selective, mechanism-based inhibitor of NEDD8-activating enzyme (NAE), another E1 enzyme, by forming a NEDD8-MLN4924 adduct that tightly binds at the active site of NAE, a novel mechanism termed substrate-assisted inhibition (Brownell, J. E., Sintchak, M. D., Gavin, J. M., Liao, H., Bruzzese, F. J., Bump, N. J., Soucy, T. A., Milhollen, M. A., Yang, X., Burkhardt, A. L., Ma, J., Loke, H. K., Lingaraj, T., Wu, D., Hamman, K. B., Spelman, J. J., Cullis, C. A., Langston, S. P., Vyskocil, S., Sells, T. B., Mallender, W. D., Visiers, I., Li, P., Claiborne, C. F., Rolfe, M., Bolen, J. B., and Dick, L. R. (2010) Mol. Cell 37, 102-111). In the present study, substrate-assisted inhibition of human UAE (Ube1) by another adenosine sulfamate analogue, 5'-O-sulfamoyl-N(6)-[(1S)-2,3-dihydro-1H-inden-1-yl]-adenosine (Compound I), a nonselective E1 inhibitor, was characterized. Compound I inhibited UAE-dependent ATP-PP(i) exchange activity, caused loss of UAE thioester, and inhibited E1-E2 transthiolation in a dose-dependent manner. Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácidos Sulfónicos/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Difosfatos/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Cinética , Compuestos de Sulfhidrilo/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo
6.
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
7.
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
8.
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
9.
Mol Cell ; 37(1): 102-11, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20129059

RESUMEN

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.


Asunto(s)
Adenosina Monofosfato/metabolismo , Ciclopentanos/metabolismo , Inhibidores Enzimáticos/metabolismo , Pirimidinas/metabolismo , Ubiquitinas/metabolismo , Adenosina Monofosfato/química , Sitios de Unión , Unión Competitiva , Línea Celular Tumoral , Cristalografía por Rayos X , Ciclopentanos/química , Ciclopentanos/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Proteína NEDD8 , Estructura Terciaria de Proteína , Pirimidinas/química , Pirimidinas/farmacología , Ubiquitinas/química
10.
Anal Biochem ; 394(1): 24-9, 2009 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-19602421

RESUMEN

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous "E1s" initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [32P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [32P]PPi and then measuring [32P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [32P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.


Asunto(s)
Adenosina Trifosfato/metabolismo , Carbón Orgánico/química , Difosfatos/metabolismo , Papel , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/química , Difosfatos/química , Humanos , Marcaje Isotópico , Cinética , Modelos Lineales , Proteína NEDD8 , Especificidad por Sustrato , Volumetría , Enzimas Activadoras de Ubiquitina/química
11.
Mol Cancer Ther ; 5(12): 3052-61, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17172407

RESUMEN

Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time- and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated.


Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Lactonas/farmacología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Pirazinas/farmacología , Pirroles/farmacología , Animales , Antineoplásicos/química , Unión Competitiva , Ácidos Borónicos/química , Bortezomib , Estabilidad de Medicamentos , Femenino , Células HT29 , Células HeLa , Humanos , Lactonas/química , Ratones , Ratones Desnudos , Ratones SCID , Inhibidores de Proteasas/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/química , Pirroles/química , Ensayos Antitumor por Modelo de Xenoinjerto
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