RESUMEN
Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing.
Asunto(s)
Antivirales , ATPasas de Translocación de Protón Vacuolares , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Ratones , Relación Estructura-Actividad , Antivirales/farmacología , Antivirales/química , Antivirales/farmacocinética , Antivirales/síntesis química , Humanos , Estructura Molecular , Ebolavirus/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Relación Dosis-Respuesta a Droga , Lignanos/farmacología , Lignanos/química , Naftalenos/farmacología , Naftalenos/química , Naftalenos/farmacocinética , Naftalenos/síntesis química , Internalización del Virus/efectos de los fármacosRESUMEN
Proliferating cell nuclear antigen (PCNA) is a homo-trimeric protein complex that clamps around DNA to tether DNA polymerases to the template during replication and serves as a hub for many other interacting proteins. It regulates DNA metabolic processes and other vital cellar functions through the binding of proteins having short linear motifs (SLiMs) like the PIP-box (PCNA-interacting protein-box) or the APIM (AlkB homolog 2 PCNA-interacting motif) in the hydrophobic pocket where SLiMs bind. However, overproducing TbPCNA or human PCNA (hPCNA) in the pathogenic protist Trypanosoma brucei triggers a dominant-negative phenotype of arrested proliferation. The mechanism for arresting T. brucei proliferation requires the overproduced PCNA orthologs to have functional intact SLiM-binding pocket. Sight-directed mutagenesis studies showed that T. brucei overproducing PCNA variants with disrupted SLiM-binding pockets grew normally. We hypothesized that chemically disrupting the SLiM-binding pocket would restore proliferation in T. brucei, overproducing PCNA orthologs. Testing this hypothesis is the proof-of-concept for a T. brucei-based PCNA screening assay. The assay design is to discover bioactive small molecules that restore proliferation in T. brucei strains that overproduce PCNA orthologs, likely by disrupting interactions in the SLiM-binding pocket. The pilot screen for this assay discovered two hit compounds that linked to predetermined PCNA targets. Compound #1, a known hPCNA inhibitor, had selective bioactivity to hPCNA overproduced in T. brucei, validating the assay. Compound #6 had promiscuous bioactivity for hPCNA and TbPCNA but is the first compound discovered with bioactivity for inhibiting TbPCNA.
Asunto(s)
Replicación del ADN , Trypanosoma brucei brucei , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Trypanosoma brucei brucei/metabolismo , ADN/metabolismo , Mutagénesis , Unión ProteicaRESUMEN
Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion into a toxic gas and access to large quantities through several industries. The high safety index of hydroxocobalamin is a cornerstone quality as a cyanide scavenger. Unfortunately, intravenous infusion of hydroxocobalamin limits the utility in a mass casualty setting. We previously reported platinum(II) [Pt(II)] complexes with trans-directing sulfur ligands as an efficacious alternative to hydroxocobalamin when delivered by a bolus intramuscular injection in mice and rabbits. Thus, to enable Pt(II) as an alternative to hydroxocobalamin, a high safety factor is needed. The objective is to maintain efficacy and mitigate the risk for nephrotoxicity. Platinum amino acid complexes with the ability to form five- or six-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy was evaulated in the zebrafish and mice cyanide exposure models. In addition, Pt(II) complex toxicity and pharmacokinetics were evaluated in a cyanide naive Sprague-Dawley model. Doses for toxicity are escalated to 5x from the efficacious dose in mice using a body surface area adjustment. The results show the carboxamide ligands display a time and pH dependence on cyanide scavenging in vitro and efficacy in vivo. Additionally, exchanging the carboxylate for carboxamide showed reduced indications of renal injury. A pharmacokinetic analysis of the larger bidentate complexes displayed rapid absorption by intramuscular administration and having similar plasma exposure. These findings point to the importance of pH and ligand structures for methionine carboxamide complexes with Pt(II).
RESUMEN
The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.
Asunto(s)
Antineoplásicos , Platino (Metal) , Ratones , Conejos , Animales , Platino (Metal)/química , Pez Cebra , Cianuros , Dimetilsulfóxido , Ligandos , Sulfuros , Antineoplásicos/farmacologíaRESUMEN
REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Éteres Fenílicos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina/química , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Cromatina/química , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Daño del ADN , Humanos , Modelos Moleculares , Proteínas Nucleares/efectos de los fármacos , Nucleotidiltransferasas/efectos de los fármacos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Unión Proteica , Conformación Proteica , Dominios Proteicos , Células Tumorales Cultivadas , Ubiquitina/efectos de los fármacos , UbiquitinaciónRESUMEN
REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin. This study demonstrates the potential utility of a small-molecule REV1 UBM2 inhibitor for preventing DDT.
Asunto(s)
Daño del ADN/efectos de los fármacos , ADN/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Piperazinas/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Sitios de Unión , Línea Celular Tumoral , Cisplatino/farmacología , ADN/efectos de la radiación , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Lisina/química , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Nucleotidiltransferasas/química , Piperazinas/síntesis química , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica/efectos de los fármacos , Ubiquitinación , Rayos UltravioletaRESUMEN
This review article discusses some insights about generating novel mechanistic inhibitors of the DNA damage response and repair (DDR) pathways by focusing on protein-protein interactions (PPIs) of the key DDR components. General requirements for PPI strategies, such as selecting the target PPI site on the basis of its functionality, are discussed first. Next, on the basis of functional rationale and biochemical feasibility to identify a PPI inhibitor, 26 PPIs in DDR pathways (BER, MMR, NER, NHEJ, HR, TLS, and ICL repair) are specifically discussed for inhibitor discovery to benefit cancer therapies using a DNA-damaging agent.
Asunto(s)
Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daño del ADN/fisiología , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Terapia Molecular Dirigida/métodos , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
BACKGROUND & AIMS: Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS: Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS: SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector ß-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and ß-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS: In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-ß-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Vía de Señalización Wnt/genética , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Colestasis/complicaciones , Dietilnitrosamina , Proteína 1 Similar a ELAV/metabolismo , Células Estrelladas Hepáticas , Humanos , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Estadificación de Neoplasias , Trasplante de Neoplasias , Células Madre Neoplásicas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Tasa de Supervivencia , Transcripción Genética , beta Catenina/metabolismo , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.
Asunto(s)
Antineoplásicos/farmacología , Reparación del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Mad2/antagonistas & inhibidores , Pirimidinonas/farmacología , Tiofenos/farmacología , Antineoplásicos/síntesis química , Cisplatino/farmacología , ADN Polimerasa Dirigida por ADN , Resistencia a Antineoplásicos , Células HeLa , Humanos , Unión Proteica , Pirimidinonas/síntesis química , Relación Estructura-Actividad , Tiofenos/síntesis químicaRESUMEN
DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP-DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP-DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.
Asunto(s)
Química Clic , Aductos de ADN/análisis , Reparación del ADN , Ficusina/química , Colorantes Fluorescentes/química , Alquinos/química , Azidas/química , Cobre/química , Células HeLa , Humanos , Rayos UltravioletaRESUMEN
Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner.
Asunto(s)
Inhibidores Enzimáticos/química , Ribonucleótido Reductasas/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Tumoral , Cisplatino/química , Ensayo Cometa , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Células HEK293 , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ubiquitinación , GemcitabinaRESUMEN
Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX-809 has been reported to facilitate the folding and trafficking of F508del-CFTR and augment its channel function. The mechanism of action of VX-809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX-809: does it bind CFTR directly in order to exert its action? We synthesized two VX-809 derivatives, ALK-809 and SUL-809, that possess an alkyne group and retain the rescue capacity of VX-809. By using Cu(I) -catalyzed click chemistry, we provide evidence that the VX-809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.
Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Benzodioxoles/química , Benzodioxoles/farmacología , Química Clic , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Aminopiridinas/síntesis química , Benzodioxoles/síntesis química , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Descubrimiento de Drogas , Células HEK293 , Humanos , Mutación , Unión ProteicaRESUMEN
For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2â¼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2â¼Ub catalytic modules such as APC11-UBCH10â¼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10â¼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2â¼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/química , ADN Helicasas/química , Proteínas de Unión al ADN/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina/química , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Cristalografía por Rayos X , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.
Asunto(s)
Compuestos Organoplatinos/química , Antígeno Nuclear de Célula en Proliferación/química , Sitios de Unión , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Polarización de Fluorescencia , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Compuestos Organoplatinos/síntesis química , Compuestos Organoplatinos/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/metabolismo , Éteres Fenílicos/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Propanolaminas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Células HeLa , Humanos , Neoplasias/genética , Éteres Fenílicos/química , Antígeno Nuclear de Célula en Proliferación/química , Propanolaminas/químicaRESUMEN
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2â¼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECTâ¼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3â¼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3â¼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001.
Asunto(s)
Lisina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Homología de Secuencia de Aminoácido , Ubiquitina/química , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5 µM inhibited cellular DNA replication at 10 µM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.
Asunto(s)
Replicación del ADN/efectos de los fármacos , ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
Abstract A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 159(3.53) to glutamic acid and isoleucine160(3.54) to arginine (A159E/I160R) in TM-III of the human ß(1)-adrenergic receptor (ß(1)-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156(3.50) in the E/DRY motif of TM-III and displaced Tyr166(3.60) in intracellular loop 2. The A159E/I160R ß(1)-AR was uncoupled from G(s) as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß(1)-AR and G(s)α. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß(1)-AR and the A159E/I160R ß(1)-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT ß(1)-AR and this effect was dependent on the activation of the G(s)-cyclic AMP-dependent protein kinase â Rap â B-raf axis. However, in cells bearing the A159E/I160R ß(1)-AR, isoproterenol failed to increase the phosphorylation of ERK(1/2). These results indicate that mutations in the G(s)α-binding pocket of the GPCR interfered with receptor coupling to G(s) and with its downstream signaling cascades.
Asunto(s)
Aminoácidos/química , Citoplasma/metabolismo , Proteínas de Unión al GTP/química , Receptores Adrenérgicos beta 1/química , Aminoácidos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Isoproterenol/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Mutación , Fosforilación/efectos de los fármacos , Conformación Proteica , Receptores Adrenérgicos beta 1/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 µm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.
Asunto(s)
Replicación del ADN/fisiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Química Farmacéutica/métodos , Cromatina/metabolismo , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Citometría de Flujo/métodos , Genes Reporteros , Células HeLa , Humanos , Concentración 50 Inhibidora , Ligandos , Microscopía Fluorescente/métodos , Conformación Molecular , Péptidos/química , Fosforilación , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes/químicaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical surfaces of epithelial cells lining airway, gut and exocrine glands, where it is responsible for transepithelial salt and water transport. Several human diseases are associated with an altered channel function of CFTR. Cystic fibrosis (CF) is caused by the loss or dysfunction of CFTR-channel activity resulting from the mutations on the gene; whereas enterotoxin-induced secretory diarrheas are caused by the hyperactivation of CFTR channel function. CFTR is a validated target for drug development to treat these diseases. Significant progress has been made in developing CFTR modulator therapy by means of high-throughput screening followed by hit-to-lead optimization. Several oral administrated investigational drugs are currently being evaluated in clinical trials for CF. Also importantly, new ideas and methodologies are emerging. Targeting CFTR-containing macromolecular complexes is one such novel approach.