RESUMEN
Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.
Asunto(s)
MicroARNs , ARN Nucleolar Pequeño , Niño , Humanos , ARN Nucleolar Pequeño/genética , Ribonucleasa III/química , MicroARNs/química , Mutación , ARN Helicasas DEAD-box/genéticaRESUMEN
Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein-protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.
Asunto(s)
Productos Biológicos , Estructura Molecular , Productos Biológicos/química , Fenol , Espectroscopía de Resonancia MagnéticaRESUMEN
Hydrocarbon stapled (HCS) peptides are a class of cross-linked α-helix mimetics. The technology relies on the use of α,α'-disubstituted alkenyl amino acids, which fully contrain the helical region to typically yield peptides with enhanced structural ordering and biological activity. Recently, monosubstituted alkenyl amino acids were disclosed for peptide stapling; however, the impact that this tether has on HCS peptide structure and activity has not yet been fully explored. By applying this HCS to the disordered peptide eIF4E-binding protein 1 (4E-BP1), we discovered that this type of tethering has a dramatic effect on olefin geometry and activity of the resultant stapled peptides, where the putative trans isomer was found to exhibit enhanced in vitro and cellular inhibitory activity against eIF4E protein-protein interactions. We further demonstrated that the metathesis catalyst used for ring-closing metathesis can influence monosubstituted HCS peptide activity, presumably through alteration of the cis/trans olefin ratio. This study represents one of the first in-depth analyses of olefin isomers of a stapled peptide and highlights an additional feature for medicinal chemistry optimization of this class of peptide-based probes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Alquenos/química , Proteínas de Ciclo Celular/química , Péptidos/química , Humanos , Modelos Moleculares , Péptidos/síntesis química , Especificidad por SustratoRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m7GpppX-cap at the 5' terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis, among other cancerous phenotypes. eIF4E is the rate-limiting translation factor, and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth toward inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work toward solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.
Asunto(s)
Factor 4E Eucariótico de Iniciación , Neoplasias , Factor 4E Eucariótico de Iniciación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ARN/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Neoplasias/tratamiento farmacológicoRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) is an RNA-binding protein that binds to the m 7 GpppX-cap at the 5' terminus of coding mRNAs to initiate cap-dependent translation. While all cells require cap-dependent translation, cancer cells become addicted to enhanced translational capacity, driving the production of oncogenic proteins involved in proliferation, evasion of apoptosis, metastasis, and angiogenesis among other cancerous phenotypes. eIF4E is the rate-limiting translation factor and its activation has been shown to drive cancer initiation, progression, metastasis, and drug resistance. These findings have established eIF4E as a translational oncogene and promising, albeit challenging, anti-cancer therapeutic target. Although significant effort has been put forth towards inhibiting eIF4E, the design of cell-permeable, cap-competitive inhibitors remains a challenge. Herein, we describe our work towards solving this long-standing challenge. By employing an acyclic nucleoside phosphonate prodrug strategy, we report the synthesis of cell-permeable inhibitors of eIF4E binding to capped mRNA to inhibit cap-dependent translation.
RESUMEN
MicroRNAs (miRNAs) are a family of small noncoding RNAs that regulate gene expression. Due to their important activity in the fine-tuning of protein translation, abnormal expression of miRNAs has been linked to many human diseases, making the targeting of miRNAs attractive as a novel therapeutic strategy. Accordingly, researchers have been heavily engaged in the discovery of small molecule modulators of miRNAs. With an interest in the identification of new chemical space for targeting miRNAs, we developed a high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay (cat-ELCCA), aimed at the discovery of small molecule ligands for pre-miR-21, a miRNA that is frequently overexpressed in human cancers. From our HTS campaign, we found that natural products, a source of many impactful human medicines, may be a promising source of potential pre-miR-21-selective maturation inhibitors. Herein we describe our first efforts in natural product inhibitor discovery leading to the identification of a depsipeptide class of natural products as RNA-binding inhibitors of Dicer-mediated miRNA processing.
RESUMEN
Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) controls the initiation of cap-dependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post-translationally modify 4E-BP1 to drive aberrant cap-dependent translation. We recently reported a site-selective chemoproteomic method for uncovering kinase-substrate interactions, and using this approach, we discovered the cyclin-dependent kinase (CDK)4 as a new 4E-BP1 kinase. Herein, we describe our extension of this work and reveal the role of CDK4 in modulating 4E-BP1 activity in the transition from mitosis to G1, thereby demonstrating a novel role for this kinase in cell cycle regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Mitosis/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Fase G1/genética , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Biosíntesis de Proteínas , Piridinas/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) has emerged as a promising cancer therapeutic target due to its role in the initiation of cap-dependent translation, a process that is accelerated during tumorigenesis. To regulate the initiation of cap-dependent translation, eIF4E participates in protein-protein interactions (PPI) with binding partners, 4E-BP1 and eIF4G, which act as an inhibitor and stimulator of translation, respectively. As both of these proteins interact with eIF4E by utilizing a short, α-helical stretch of amino acids, our laboratory has been working to develop helical mimetics of these proteins, in particular 4E-BP1, to inhibit eIF4E PPIs. Herein, we describe our continued efforts in this area and report the development and characterization of a cell-penetrant lactam stapled peptide for targeting cellular eIF4E.
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Factor 4E Eucariótico de Iniciación/metabolismo , Lactamas/química , Línea Celular Tumoral , Humanos , Terapia Molecular Dirigida , Unión Proteica/efectos de los fármacos , Biosíntesis de ProteínasRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) is a key player in the initiation of cap-dependent translation through recognition of the m7GpppX cap at the 5' terminus of coding mRNAs. As eIF4E overexpression has been observed in a number of human diseases, most notably cancer, targeting this oncogenic translation initiation factor has emerged as a promising strategy for the development of novel anti-cancer therapeutics. Toward this end, in the present study, we have rationally designed a series of Bn7GxP-based PROTACs for the targeted degradation of eIF4E. Herein we describe our synthetic efforts, in addition to biochemical and cellular characterization of these compounds.
Asunto(s)
Diseño de Fármacos , Factor 4E Eucariótico de Iniciación/metabolismo , Guanosina/análogos & derivados , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Química Sintética , Factor 4E Eucariótico de Iniciación/química , Guanosina/síntesis química , Guanosina/química , Guanosina/farmacología , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Recent estimates of the human proteome suggest there are â¼20,000 protein-coding genes, the protein products of which contain >145,000 phosphosites. Unfortunately, in-depth examination of the human phosphoproteome has outpaced the ability to annotate the kinases that mediate these post-translational modifications. To obtain actionable information about phosphorylation-driven signaling cascades, it is essential to identify the kinases responsible for phosphorylating sites that differ across disease states. To fill in these gaps we have developed an unbiased, chemoproteomic approach for identifying high-confidence kinase-substrate interactions with phosphosite specificity. Using this assay, we uncovered the role of cyclin-dependent kinase 4 (CDK4), a clinically validated kinase important for cell-cycle progression, in regulating cap-dependent translation via phosphorylation of the tumor suppressor 4E-BP1. The discovery of this signaling axis sheds light on the mechanisms by which CDK4/6 inhibitors control cell proliferation and constitutes a successful example of kinase discovery using an activity-based, kinase-directed probe.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Quinasa 4 Dependiente de la Ciclina/genética , Femenino , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Persona de Mediana Edad , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein-protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Ciclo Celular/química , Diseño de Fármacos , Factor 4E Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , Fragmentos de Péptidos/síntesis química , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4E Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4G Eucariótico de Iniciación/genética , Humanos , Concentración 50 Inhibidora , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Plásmidos , Unión ProteicaRESUMEN
In a high-throughput screening campaign, we recently discovered the rRNA-binding tetracyclines, methacycline and meclocycline, as inhibitors of Dicer-mediated processing of microRNAs. Herein, we describe our biophysical and biochemical characterization of these compounds. Interestingly, although direct, albeit weak, binding to the pre-microRNA hairpins was observed, the inhibitory activity of these compounds was not due to RNA binding. Through additional biochemical and chemical studies, we revealed that metal chelation likely plays a principle role in their mechanism of inhibition. By exploring the activity of other known RNA-binding scaffolds, we identified additional disconnections between direct RNA interaction and inhibition of Dicer processing. Thus, the results presented within provide a valuable case study in the complexities of targeting RNA with small molecules, particularly with weak binding and potentially promiscuous scaffolds.
RESUMEN
Human biology is regulated by a complex network of protein-protein interactions (PPIs), and disruption of this network has been implicated in many diseases. However, the targeting of PPIs remains a challenging area for chemical probe and drug discovery. Although many methodologies have been put forth to facilitate these efforts, new technologies are still needed. Current biochemical assays for PPIs are typically limited to motif-domain and domain-domain interactions, and assays that will enable the screening of full-length protein systems, which are more biologically relevant, are sparse. To overcome this barrier, we have developed a new assay technology, "PPI catalytic enzyme-linked click chemistry assay" or PPI cat-ELCCA, which utilizes click chemistry to afford catalytic signal amplification. To validate this approach, we have applied PPI cat-ELCCA to the eIF4E-4E-BP1 and eIF4E-eIF4G PPIs, key regulators of cap-dependent mRNA translation. Using these examples, we have demonstrated that PPI cat-ELCCA is amenable to full-length proteins, large (>200 kDa) and small (â¼12 kDa), and is readily adaptable to automated high-throughput screening. Thus, PPI cat-ELCCA represents a powerful new tool in the toolbox of assays available to scientists interested in the targeting of disease-relevant PPIs.