RESUMEN
In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.
Asunto(s)
ARN Helicasas , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , ARN Helicasas/genética , ARN Helicasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Biosíntesis de Proteínas , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Ribosomas/metabolismoRESUMEN
Inhibition of eukaryotic translation initiation through unscheduled RNA clamping of the DEAD-box (DDX) RNA helicases eIF4A1 and eIF4A2 has been documented for pateamine A (PatA) and rocaglates-two structurally different classes of compounds that share overlapping binding sites on eIF4A. Clamping of eIF4A to RNA causes steric blocks that interfere with ribosome binding and scanning, rationalizing the potency of these molecules since not all eIF4A molecules need to be engaged to elicit a biological effect. In addition to targeting translation, PatA and analogs have also been shown to target the eIF4A homolog, eIF4A3-a helicase necessary for exon junction complex (EJC) formation. EJCs are deposited on mRNAs upstream of exon-exon junctions and, when present downstream from premature termination codons (PTCs), participate in nonsense-mediated decay (NMD), a quality control mechanism aimed at preventing the production of dominant-negative or gain-of-function polypeptides from faulty mRNA transcripts. We find that rocaglates can also interact with eIF4A3 to induce RNA clamping. Rocaglates also inhibit EJC-dependent NMD in mammalian cells, but this does not appear to be due to induced eIF4A3-RNA clamping, but rather a secondary consequence of translation inhibition incurred by clamping eIF4A1 and eIF4A2 to mRNA.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , ARN , Animales , ARN/metabolismo , ARN Mensajero/metabolismo , Codón sin Sentido , Exones , Factor 4A Eucariótico de Iniciación/química , Mamíferos/genéticaRESUMEN
The PI3K/Akt/mTOR kinase pathway is extensively deregulated in human cancers. One critical node under regulation of this signaling axis is eukaryotic initiation factor (eIF) 4F, a complex involved in the control of translation initiation rates. eIF4F-dependent addictions arise during tumor initiation and maintenance due to increased eIF4F activity-generally in response to elevated PI3K/Akt/mTOR signaling flux. There is thus much interest in exploring eIF4F as a small molecule target for the development of new anticancer drugs. The DEAD-box RNA helicase, eIF4A, is an essential subunit of eIF4F, and several potent small molecules (rocaglates, hippuristanol, pateamine A) affecting its activity have been identified and shown to demonstrate anticancer activity in vitro and in vivo in preclinical models. Recently, a number of new small molecules have been reported as having the capacity to target and inhibit eIF4A. Here, we undertook a comparative analysis of their biological activity and specificity relative to the eIF4A inhibitor, hippuristanol.
Asunto(s)
Antineoplásicos/química , Factor 4A Eucariótico de Iniciación/química , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Esteroles/química , Antineoplásicos/farmacología , Benzofuranos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Compuestos Epoxi/química , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación/química , Humanos , Macrólidos/química , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Esteroles/farmacología , Serina-Treonina Quinasas TOR/genética , Tiazoles/químicaRESUMEN
Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eµ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e∆38 fs/+/Eµ-Myc or Eif4a1∆5 fs/+/Eµ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Factor 4F Eucariótico de Iniciación/genética , Animales , Femenino , Regulación de la Expresión Génica/genética , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subunidades de Proteína/genética , Espermatogénesis/genéticaRESUMEN
Eukaryotic cellular mRNAs possess a 5' cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/genética , Biosíntesis de Proteínas , Secuencia de Aminoácidos/genética , Animales , Factor 4E Eucariótico de Iniciación/química , Factor 4F Eucariótico de Iniciación/química , Células HEK293 , Humanos , Ratones , Unión Proteica/genética , Análogos de Caperuza de ARN/genética , Caperuzas de ARN/genética , ARN Mensajero/genética , Ribosomas/genéticaRESUMEN
We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.
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Interferencia de ARN , ARN Interferente Pequeño/química , Proteínas Argonautas/metabolismo , Conformación de Carbohidratos , Células HeLa , Humanos , Luciferasas de Luciérnaga/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/inmunología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.
Asunto(s)
Regiones no Traducidas 5' , Resistencia a Antineoplásicos/genética , Factor 4A Eucariótico de Iniciación/genética , Esteroles/farmacología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Mutación , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.
Asunto(s)
Aglaia/química , Antimaláricos/administración & dosificación , Malaria Cerebral/tratamiento farmacológico , Extractos Vegetales/administración & dosificación , Animales , Modelos Animales de Enfermedad , Eritrocitos/parasitología , Factor 4F Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eµ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.
Asunto(s)
Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas Genéticas , Animales , Proteínas Asociadas a CRISPR/metabolismo , Femenino , Marcación de Gen , Genes p53/genética , Genoma/genética , Mutación INDEL/genética , Estimación de Kaplan-Meier , Lentivirus/genética , Linfoma/genética , Linfoma/mortalidad , Linfoma/terapia , Ratones , Ratones Endogámicos C57BL , Mutación , Reproducibilidad de los ResultadosRESUMEN
IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caperuzas de ARN/química , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Humanos , Metilación , Ratones , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , Posición Específica de Matrices de Puntuación , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Relación Estructura-ActividadRESUMEN
All cellular cytoplasmic mRNAs are capped at their 5' ends with an m7GpppN group. Several proteins that mediate cap function have been identified by cap affinity purification, enabling their characterization in a number of biological processes. Among these, eukaryotic initiation factor (eIF) 4E is the best characterized and plays a critical role in regulating ribosome recruitment to mRNAs during translation initiation. Cap affinity chromatography is often used to identify eIF4E-interacting proteins, which could play critical roles in molding the eIF4E-interactome and impacting on eIF4E-directed translation. Here we address how improper implementation of this technology can lead to false conclusions and provide recommendations to ensure correct interpretation of data obtained by this approach.
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Cromatografía de Afinidad/métodos , Factor 4E Eucariótico de Iniciación/aislamiento & purificación , Error Científico Experimental , Escherichia coli , Células HeLa , Humanos , Unión Proteica , Caperuzas de ARN/química , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Sefarosa/químicaRESUMEN
The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.
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Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , Genes MDR/genética , Genes MDR/fisiología , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Nucleósidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidadRESUMEN
Synthesis of aza-rocaglates, nitrogen-containing analogues of the rocaglate natural products, is reported. The route features ESIPT-mediated (3+2) photocycloaddition of 1-alkyl-2-aryl-3-hydroxyquinolinones with the dipolarophile methyl cinnamate. A continuous photoflow reactor was utilized for photocycloadditions. An array of compounds bearing the hexahydrocyclopenta[b]indole core structure was synthesized and evaluated in translation inhibition assays.
RESUMEN
BACKGROUND: Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established. METHODS: Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed. RESULTS: We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model. CONCLUSIONS: Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Linfoma/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Pirroles/administración & dosificación , Animales , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Sinergismo Farmacológico , Humanos , Indoles , Linfoma/metabolismo , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Pirroles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DExH/D box proteins are molecular motors that utilize the energy derived from NTP hydrolysis to perform work - from helicases that remodel RNA to RNPases that alter RNA-protein complexes. Members of this class of proteins are uniquely placed along the RNA information highway to regulate the flow of genetic information. They have been implicated in a number of nodal points encompassing nuclear, cytoplasmic, and organellar RNA-based processes. The identification and characterization of three unique natural products that selectively inhibit the activity of eukaryotic initiation factor (eIF)4A (DDX2) has provided proof-of-principle that the activity of DExH/D box family members can be selectively targeted. Extending these achievements to other DExH/D box proteins is an important future challenge for drugging this family of proteins. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.
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ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/genética , Haplorrinos , Humanos , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismo , Alineación de SecuenciaRESUMEN
The recruitment of ribosomes to eukaryotic cellular mRNAs requires the activity of two prototypic RNA helicases, eukaryotic initiation factor (eIF) 4AI and eIF4AII. The eIF4A isoforms are highly conserved, are thought to be functionally interchangeable, and are directed to the 5' m(7)GpppN cap structure of mRNAs during translation initiation by virtue of their assembly into eIF4F, a heterotrimeric complex that also harbors the eIF4E cap binding protein and eIF4G scaffolding unit. During the course of RNA interference experiments aimed at investigating the respective roles of eIF4AI and eIF4AII in translation, we uncovered a cellular response pathway whereby suppression of eIF4AI increases transcription of the eIF4AII gene, leading to elevated eIF4AII mRNA and protein levels. Inhibition of eIF4AI suppresses protein synthesis, and although eIF4AII protein levels increase above and beyond what should be sufficient to compensate for the decrease in eIF4AI levels, there is no corresponding rescue of translation or of the block on cellular proliferation that occurs upon eIF4AI suppression. These results were phenocopied using the small molecule eIF4A inhibitor hippuristanol. Taken together, our results indicate that eIF4AI and eIF4AII expression appear linked and that the two protein isoforms exhibit functional differences.
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Factor 4A Eucariótico de Iniciación/metabolismo , Transcripción Genética/fisiología , Adulto , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Femenino , Feto/efectos de los fármacos , Feto/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Próstata/efectos de los fármacos , Próstata/metabolismo , Isoformas de Proteínas/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Esteroles/farmacología , Testículo/efectos de los fármacos , Testículo/metabolismo , Timo/efectos de los fármacos , Timo/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Despite the growing number of examples of small-molecule inhibitors that disrupt protein-protein interactions (PPIs), the origin of druggability of such targets is poorly understood. To identify druggable sites in protein-protein interfaces we combine computational solvent mapping, which explores the protein surface using a variety of small "probe" molecules, with a conformer generator to account for side-chain flexibility. Applications to unliganded structures of 15 PPI target proteins show that the druggable sites comprise a cluster of binding hot spots, distinguishable from other regions of the protein due to their concave topology combined with a pattern of hydrophobic and polar functionality. This combination of properties confers on the hot spots a tendency to bind organic species possessing some polar groups decorating largely hydrophobic scaffolds. Thus, druggable sites at PPI are not simply sites that are complementary to particular organic functionality, but rather possess a general tendency to bind organic compounds with a variety of structures, including key side chains of the partner protein. Results also highlight the importance of conformational adaptivity at the binding site to allow the hot spots to expand to accommodate a ligand of drug-like dimensions. The critical components of this adaptivity are largely local, involving primarily low energy side-chain motions within 6 Å of a hot spot. The structural and physicochemical signature of druggable sites at PPI interfaces is sufficiently robust to be detectable from the structure of the unliganded protein, even when substantial conformational adaptation is required for optimal ligand binding.
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Descubrimiento de Drogas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Sitios de Unión , Ligandos , Sondas Moleculares , Unión Proteica , Conformación Proteica/efectos de los fármacos , Proteínas/metabolismo , SolventesRESUMEN
Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.
Asunto(s)
Benzoatos/farmacología , Factor 4F Eucariótico de Iniciación/antagonistas & inhibidores , Linfoma/metabolismo , Modelos Moleculares , Biosíntesis de Proteínas/fisiología , Animales , Western Blotting , Factor 4F Eucariótico de Iniciación/biosíntesis , Factor 4G Eucariótico de Iniciación/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Linfoma/tratamiento farmacológico , Ratones , Unión ProteicaRESUMEN
Coronaviruses are highly transmissible respiratory viruses that cause symptoms ranging from mild congestion to severe respiratory distress. The recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need for new antivirals with broad-acting mechanisms to combat increasing emergence of new variants. Currently, there are only a few antivirals approved for treatment of SARS-CoV-2. Previously, the rocaglate natural product silvestrol and synthetic rocaglates such as CR-1-31b were shown to have antiviral effects by inhibiting eukaryotic translation initiation factor 4A1 (eIF4A) function and virus protein synthesis. In this study, we evaluated amidino-rocaglates (ADRs), a class of synthetic rocaglates with the most potent eIF4A-inhibitory activity to-date, for inhibition of SARS-CoV-2 infection. This class of compounds showed low nanomolar potency against multiple SARS-CoV-2 variants and in multiple cell types, including human lung-derived cells, with strong inhibition of virus over host protein synthesis and low cytotoxicity. The most potent ADRs were also shown to be active against two highly pathogenic and distantly related coronaviruses, SARS-CoV and MERS-CoV. Mechanistically, cells with mutations of eIF4A1, which are known to reduce rocaglate interaction displayed reduced ADR-associated loss of cellular function, consistent with targeting of protein synthesis. Overall, ADRs and derivatives may offer new potential treatments for SARS-CoV-2 with the goal of developing a broad-acting anti-coronavirus agent.
RESUMEN
Glioblastoma (GBM) is the most aggressive and frequently occurring type of malignant brain tumor in adults. The initiation, progression, and recurrence of malignant tumors are known to be driven by a small subpopulation of cells known as tumor-initiating cells or cancer stem cells (CSCs). GBM CSCs play a pivotal role in orchestrating drug resistance and tumor relapse. As a prospective avenue for GBM intervention, the targeted suppression of GBM CSCs holds considerable promise. In this study, we found that rocaglates, compounds which are known to inhibit translation via targeting of the DEAD-box helicase eIF4A, exert a robust, dose-dependent cytotoxic impact on GBM CSCs with minimal killing of nonstem GBM cells. Subsequent optimization identified novel rocaglate derivatives (rocaglate acyl sulfamides or Roc ASFs) that selectively inhibit GBM CSCs with nanomolar EC50 values. Furthermore, comparative evaluation of a lead CSC-optimized Roc ASF across diverse mechanistic and target profiling assays revealed suppressed translation inhibition relative to that of other CSC-selective rocaglates, with enhanced targeting of the DEAD-box helicase DDX3X, a recently identified secondary target of rocaglates. Overall, these findings suggest a promising therapeutic strategy for targeting GBM CSCs.