RESUMEN
Features from a high throughput screening (HTS) hit and a previously reported scaffold were combined to generate 1,7-naphthyridones as novel KDM5 enzyme inhibitors with nanomolar potencies. These molecules exhibited high selectivity over the related KDM4C and KDM2B isoforms. An X-ray co-crystal structure of a representative molecule bound to KDM5A showed that these inhibitors are competitive with the co-substrate (2-oxoglutarate or 2-OG).
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Naftiridinas/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Humanos , Células de Riñón Canino Madin Darby , Naftiridinas/química , Relación Estructura-ActividadRESUMEN
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteína 2 de Unión a Retinoblastoma/metabolismo , Relación Estructura-ActividadRESUMEN
Levels of residual structure in disordered interaction domains determine in vitro binding affinities, but whether they exert similar roles in cells is not known. Here, we show that increasing residual p53 helicity results in stronger Mdm2 binding, altered p53 dynamics, impaired target gene expression and failure to induce cell cycle arrest upon DNA damage. These results establish that residual structure is an important determinant of signaling fidelity in cells.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteínas Proto-Oncogénicas c-mdm2/química , Transducción de Señal , Proteína p53 Supresora de Tumor/química , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Daño del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Rayos gamma , Regulación de la Expresión Génica , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Mutación , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by â¼60- to 2000-fold at 1-2 µM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.
Asunto(s)
Compuestos de Bifenilo/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia/tratamiento farmacológico , Nitrofenoles/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Animales , Resistencia a Antineoplásicos/genética , Células HL-60 , Humanos , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Pirroles , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Oxygen-evolving photosynthetic organisms regulate carbon metabolism through a light-dependent redox signalling pathway. Electrons are shuttled from photosystem I by means of ferredoxin (Fdx) to ferredoxin-thioredoxin reductase (FTR), which catalyses the two-electron-reduction of chloroplast thioredoxins (Trxs). These modify target enzyme activities by reduction, regulating carbon flow. FTR is unique in its use of a [4Fe-4S] cluster and a proximal disulphide bridge in the conversion of a light signal into a thiol signal. We determined the structures of FTR in both its one- and its two-electron-reduced intermediate states and of four complexes in the pathway, including the ternary Fdx-FTR-Trx complex. Here we show that, in the first complex (Fdx-FTR) of the pathway, the Fdx [2Fe-2S] cluster is positioned suitably for electron transfer to the FTR [4Fe-4S] centre. After the transfer of one electron, an intermediate is formed in which one sulphur atom of the FTR active site is free to attack a disulphide bridge in Trx and the other sulphur atom forms a fifth ligand for an iron atom in the FTR [4Fe-4S] centre--a unique structure in biology. Fdx then delivers a second electron that cleaves the FTR-Trx heterodisulphide bond, which occurs in the Fdx-FTR-Trx complex. In this structure, the redox centres of the three proteins are aligned to maximize the efficiency of electron transfer from the Fdx [2Fe-2S] cluster to the active-site disulphide of Trxs. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme and describe previously unknown interaction networks for both Fdx and Trx (refs 4-6).
Asunto(s)
Ferredoxinas/química , Proteínas Hierro-Azufre/química , Oxidorreductasas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Transporte de Electrón , Ferredoxinas/metabolismo , Hierro/química , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Proteínas Recombinantes , Spinacia oleracea , Relación Estructura-Actividad , Synechocystis , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.
Asunto(s)
Fosforribosilglicinamida-Formiltransferasa/química , Fosforribosilglicinamida-Formiltransferasa/genética , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosforribosilglicinamida-Formiltransferasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por SustratoRESUMEN
A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxin/Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxin-Trx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.
Asunto(s)
Ferredoxinas/fisiología , Proteínas de Plantas/análisis , Plastidios/metabolismo , Almidón/metabolismo , Tiorredoxinas/metabolismo , Triticum/metabolismo , Aminoácidos/biosíntesis , Proteínas Hierro-Azufre , Lípidos/biosíntesis , Nucleótidos/biosíntesis , Oxidorreductasas/metabolismo , Fotosíntesis , Proteínas de Plantas/metabolismo , Pliegue de Proteína , Proteómica , Semillas/metabolismoRESUMEN
Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.
Asunto(s)
Disulfuros/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxidorreductasas/metabolismo , Análisis Espectral/métodos , Sitios de Unión , Catálisis , Cisteína/química , Cisteína/metabolismo , Disulfuros/química , Electrones , Proteínas Hierro-Azufre/química , Ligandos , Modelos Químicos , Oxidación-Reducción , Oxidorreductasas/químicaRESUMEN
Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.
Asunto(s)
Proteínas Bacterianas/análisis , Cianobacterias/enzimología , Oxidorreductasas/análisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genéticaRESUMEN
Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.
Asunto(s)
Peroxidasas/metabolismo , Populus/metabolismo , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Secuencia de Bases , Dominio Catalítico/genética , Tiorredoxinas en Cloroplasto , Cloroplastos/metabolismo , ADN de Plantas/genética , Dimerización , Transporte de Electrón , Expresión Génica , Genes de Plantas , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Peroxidasas/química , Peroxidasas/genética , Peroxirredoxinas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/genética , Populus/microbiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Mitochondria contain thioredoxin (Trx), a regulatory disulfide protein, and an associated flavoenzyme, NADP/Trx reductase, which provide a link to NADPH in the organelle. Unlike animal and yeast counterparts, the function of Trx in plant mitochondria is largely unknown. Accordingly, we have applied recently devised proteomic approaches to identify soluble Trx-linked proteins in mitochondria isolated from photosynthetic (pea and spinach leaves) and heterotrophic (potato tubers) sources. Application of the mitochondrial extracts to mutant Trx affinity columns in conjunction with proteomics led to the identification of 50 potential Trx-linked proteins functional in 12 processes: photorespiration, citric acid cycle and associated reactions, lipid metabolism, electron transport, ATP synthesis/transformation, membrane transport, translation, protein assembly/folding, nitrogen metabolism, sulfur metabolism, hormone synthesis, and stress-related reactions. Almost all of these targets were also identified by a fluorescent gel electrophoresis procedure in which reduction by Trx can be observed directly. In some cases, the processes targeted by Trx depended on the source of the mitochondria. The results support the view that Trx acts as a sensor and enables mitochondria to adjust key reactions in accord with prevailing redox state. These and earlier findings further suggest that, by sensing redox in chloroplasts and mitochondria, Trx enables the two organelles of photosynthetic tissues to communicate by means of a network of transportable metabolites such as dihydroxyacetone phosphate, malate, and glycolate. In this way, light absorbed and processed by means of chlorophyll can be perceived and function in regulating fundamental mitochondrial processes akin to its mode of action in chloroplasts.
Asunto(s)
Mitocondrias/metabolismo , Tiorredoxinas/metabolismo , Apoptosis , Cromatografía de Afinidad , Metabolismo Energético , Enzimas/metabolismo , Oxidación-Reducción , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Spinacia oleracea/citología , Spinacia oleracea/enzimología , Spinacia oleracea/metabolismoRESUMEN
Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.
Asunto(s)
Cloroplastos/genética , Proteómica , Tiorredoxinas/genética , Clonación Molecular , Oscuridad , Enzimas/metabolismo , Luz , Oxidación-Reducción , Proteínas de Plantas/biosíntesis , Proteínas Recombinantes/metabolismo , Spinacia oleracea/genética , Spinacia oleracea/metabolismo , Tiorredoxinas/metabolismoRESUMEN
Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe-4S] in close proximity to a redox active disulfide. Mössbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe-4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the N-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe-4S]2+ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe-4S]3+ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe-S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe-S enzymes.