RESUMEN
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Asunto(s)
Antimaláricos , Malaria Falciparum , Terapia Molecular Dirigida , Plasmodium falciparum , Biosíntesis de Proteínas , Proteínas Protozoarias , Tirosina-ARNt Ligasa , Adenosina/análogos & derivados , Animales , Antimaláricos/química , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Cristalografía por Rayos X , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Ratones , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Biosíntesis de Proteínas/efectos de los fármacos , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Ácidos Sulfónicos/química , Tirosina-ARNt Ligasa/química , Tirosina-ARNt Ligasa/metabolismoRESUMEN
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Asunto(s)
Compuestos de Boro/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Administración Oral , Animales , Compuestos de Boro/administración & dosificación , Compuestos de Boro/química , Dominio Catalítico , Humanos , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Plasmodium falciparum/enzimología , Inhibidores de Proteasoma/administración & dosificación , Inhibidores de Proteasoma/químicaRESUMEN
Introduction: The proteasome is a multi-subunit enzyme complex responsible for the turnover of short-lived, abnormal or damaged proteins in eukaryotic cells. As organisms that undergo rapid growth and cell division, protozoan parasites exist on the knife-edge of proteotoxic catastrophe and thus rely heavily on their protein quality control machinery for survival. Because of this, the proteasome has recently emerged as a desirable drug target.Area covered: This review focuses on efforts to identify protozoan parasite-specific proteasome inhibitors using substrate profiling, library screening, and in vitro evolution of resistance approaches to inform medicinal chemistry. Targeting the parasite's 20S proteasome chymotrypsin-like (ß5) activity and selectively inhibiting protein turnover in parasites compared to human cells are critical properties of potent, selective inhibitors.Expert opinion: Proteasome inhibitors have the potential for rapid action against all stages, all species and all strains of plasmodium and kinetoplastid parasites. Given the high level of conservation of proteasome active sites in eukaryotes, an important challenge is achieving inhibitors that show sufficient selectivity while maintaining properties consistent with drug development.
Asunto(s)
Antiprotozoarios/farmacología , Inhibidores de Proteasoma/farmacología , Infecciones por Protozoos/tratamiento farmacológico , Animales , Desarrollo de Medicamentos , Humanos , Parásitos/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Infecciones por Protozoos/parasitologíaRESUMEN
The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.
Asunto(s)
Plasmodium falciparum/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Artemisininas/farmacología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Multimerización de Proteína , Proteostasis , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
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/químicaRESUMEN
Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.
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 XenoinjertoRESUMEN
Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Sumoilación , Proliferación Celular/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc , Humanos , Mitosis/efectos de los fármacos , Neoplasias/genética , Neoplasias/patología , Procesamiento Proteico-Postraduccional , Células Tumorales CultivadasRESUMEN
The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met â« Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro â« Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Borónicos/química , Complejos Multienzimáticos/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Oligopéptidos/química , Biblioteca de Péptidos , Inhibidores de Serina Proteinasa/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ácidos Borónicos/farmacología , Relación Dosis-Respuesta a Droga , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Oligopéptidos/farmacología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
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 Uba5â¼Ufm1 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 Uba5â¼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5â¼Ufm1 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 Uba5â¼Ufm1 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/metabolismoRESUMEN
Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The discovery of MLN4924 (Brownell et al., Mol Cell 37: 102-111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrometry-based method to quantify E1 and Ubls using isotope-labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8-MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentration represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition.
Asunto(s)
Ciclopentanos/química , Pirimidinas/química , Enzimas Activadoras de Ubiquitina/química , Ubiquitinas/química , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Femenino , Células HCT116 , Células HeLa , Humanos , Marcaje Isotópico , Proteína NEDD8 , Nanotecnología , Péptidos/análisis , Ratas , Ratas Desnudas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masas en Tándem , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
Cellular effects of a Nedd8-activating enzyme (NAE) inhibitor, MLN4924, using the AlphaScreen format were explored. MLN4924 acts as a substrate-assisted inhibitor of NAE by forming a tight binding Nedd8-MLN4924 adduct. The inhibited enzyme can no longer transfer Nedd8 downstream to modify and activate the E3 cullin-RING ligases. This results in the stabilization of proteins regulated by the proteasome, leading to cell death. These studies monitored the endogenous cellular changes to NAEâ¼Nedd8 thioester, the formation of the Nedd8-MLN4924 adduct, and the reduction in the Cul1-Nedd8. Lysates derived from MLN4924-treated HCT116 cells showed that whereas the ß-subunit of NAE remained constant, reductions of both NAEâ¼Nedd8 thioester and Cul1-Nedd8 levels occurred with a concomitant rise of the adduct. Moreover, the formation of the Nedd8-MLN4924 adduct was approximately stoichiometric with the concentration of NAEß. Higher density 384-well cell-based assays illustrated the kinetics of enzyme inactivation across a wider range of MLN4924 concentrations, showing a rapid loss of NAEâ¼Nedd8 thioester and Cul1-Nedd8. The reduction of NAEâ¼Nedd8 thioester precedes the loss of Cul1-Nedd8 at twice the rate. Finally, these results clearly demonstrate the utility of the homogeneous assay for quantitative assessment of these endogenous cellular components in a 384-well plate in response to inhibition of NAE by MLN4924.
Asunto(s)
Ciclopentanos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Células HCT116 , Humanos , Unión Proteica , Subunidades de Proteína , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
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 XenoinjertoRESUMEN
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/genéticaRESUMEN
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Ubiquitinas/metabolismo , Animales , Ácidos Borónicos/farmacología , Bortezomib , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Inhibidores Enzimáticos/farmacología , Células HEK293 , Células HeLa , Humanos , Proteína NEDD8 , Pirazinas/farmacología , Transfección , Ubiquitina/análisis , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , Ubiquitinas/análisis , Ubiquitinas/genéticaRESUMEN
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/metabolismoRESUMEN
The ubiquitin-proteasome system (UPS) and ubiquitin-like protein (UBL) conjugation pathways are integral to cellular protein homeostasis. The growing recognition of the fundamental importance of these pathways to normal cell function and in disease has prompted an in-depth search for small-molecule inhibitors that selectively block the function of these pathways. However, our limited understanding of the molecular mechanisms and biological consequences of UBL conjugation is a significant hurdle to identifying drug-like inhibitors of enzyme targets within these pathways. Here, we highlight recent advances in understanding the role of some of these enzymes and how these new insights may be the key to developing novel therapeutics for diseases including immuno-inflammatory disorders, cancer, infectious diseases, cardiovascular disease and neurodegenerative disorders.
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Sistemas de Liberación de Medicamentos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , HumanosRESUMEN
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 MolecularesRESUMEN
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/metabolismoRESUMEN
MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.