RESUMO
Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Fase S/efeitos dos fármacos , Células HL-60 , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.
Assuntos
Autofagossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Biossíntese de Proteínas , Animais , Autofagia/fisiologia , Células Cultivadas , HumanosRESUMO
Onions can be damaged by Fusarium basal rot caused by the soilborne fungus Fusarium oxysporum f. sp. cepae (FOC). Control of this pathogen is challenging since there is limited genetic resistance in onion. The identification of molecules that inhibit this pathogen is needed. Antagonism screening showed Brevibacillus fortis NRS-1210 secreted antifungal compounds into growth medium. The spent growth medium, diluted 1:1, inhibited growth of FOC conidia after seven hours and killed 67-91% of conidia after 11 h. The spent medium also inhibited growth of propagules from F. graminearum, F. proliferatum, F. verticillioides and Galactomyces citri-aurantii. Full strength spent growth medium did not effectively kill FOC conidia and chlamydospores inoculated into a sand cornmeal mixture. In silico analysis of the B. fortis NRS-1210 genome indicated the biosynthetic clusters of several antibiotics. Fractionation of spent medium followed by reverse-phase liquid chromatography with tandem mass spectrometry analysis found that fractions with the most antifungal activity contained a combination of edeines A, B and F and no other recognized antibiotics. 1H NMR signals of the active fraction corresponded to edeine, a pentapeptide with broad spectrum antimicrobial activity which blocks translation in both prokaryotes and eukaryotes. Comparative genomics of Brevibacillus genomes shows edeine producers form a clade which consists of: Brevibacillus brevis, Brevibacillus formosus, 'Brevibacillus antibioticus', Brevibacillus schisleri, Brevibacillus fortis, and Brevibacillus porteri. This observation suggests edeine played an important role in the evolution and speciation of the Brevibacillus genus.
Assuntos
Brevibacillus/metabolismo , Edeína/biossíntese , Edeína/farmacologia , Fusarium/efeitos dos fármacos , Cebolas/microbiologia , Doenças das Plantas/prevenção & controle , Esporos Fúngicos/efeitos dos fármacos , Antifúngicos/farmacologia , Brevibacillus/classificação , Brevibacillus/genética , Edeína/química , Genoma Bacteriano/genética , Filogenia , Doenças das Plantas/microbiologia , Saccharomycetales/efeitos dos fármacos , Metabolismo Secundário/genéticaRESUMO
In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidinâ S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.
Assuntos
Antituberculosos/química , Desenho de Fármacos , Peptídeos/química , Piranos/química , Animais , Antituberculosos/farmacologia , Chlorocebus aethiops , Cristalografia por Raios X , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleosídeos de Pirimidina/química , Células THP-1 , Thermus thermophilus/química , Células VeroRESUMO
Synthetic analogs of the 5' end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC50 similar to dinucleotide triphosphate m(7)GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m(7)GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity.
Assuntos
Fosfatos/química , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Análogos de Capuz de RNA/farmacologia , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Triazóis/química , Animais , Análogos de Capuz de RNA/síntese química , Coelhos , Reticulócitos/citologia , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Globinas beta/genética , Globinas beta/metabolismoRESUMO
The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-Å crystal structure of BlaS bound to a 70Sâ tRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3' terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Nucleosídeos/farmacologia , RNA de Transferência/química , Thermus thermophilus/metabolismoRESUMO
We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/metabolismo , Antivirais/síntese química , Antivirais/química , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.
Assuntos
Antivirais/farmacologia , Benzoxazóis/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Ribossomos/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Benzoxazóis/síntese química , Benzoxazóis/química , Hepacivirus/química , Ligantes , Modelos Moleculares , Estrutura Molecular , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.
RESUMO
The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.
Assuntos
Antifúngicos , Fatores de Iniciação em Eucariotos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Antifúngicos/farmacologia , Mutação , Biossíntese de Proteínas/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Substituição de AminoácidosRESUMO
Candida species are among the most prevalent causes of systemic fungal infections, which account for â¼1.5 million annual fatalities. Here, we build on a compound screen that identified the molecule N-pyrimidinyl-ß-thiophenylacrylamide (NP-BTA), which strongly inhibits Candida albicans growth. NP-BTA was hypothesized to target C. albicans glutaminyl-tRNA synthetase, Gln4. Here, we confirmed through in vitro amino-acylation assays NP-BTA is a potent inhibitor of Gln4, and we defined how NP-BTA arrests Gln4's transferase activity using co-crystallography. This analysis also uncovered Met496 as a critical residue for the compound's species-selective target engagement and potency. Structure-activity relationship (SAR) studies demonstrated the NP-BTA scaffold is subject to oxidative and non-oxidative metabolism, making it unsuitable for systemic administration. In a mouse dermatomycosis model, however, topical application of the compound provided significant therapeutic benefit. This work expands the repertoire of antifungal protein synthesis target mechanisms and provides a path to develop Gln4 inhibitors.
Assuntos
Aminoacil-tRNA Sintetases , Antifúngicos , Animais , Camundongos , Antifúngicos/farmacologia , Aminoacil-tRNA Sintetases/genética , Candida albicans , Relação Estrutura-AtividadeRESUMO
tRNA methyltransferase 9 (Trm9)-catalysed tRNA modifications have been shown to translationally enhance the DNA damage response (DDR). Here, we show that Saccharomyces cerevisiae trm9Δ, distinct DNA repair and spindle assembly checkpoint (SAC) mutants are differentially sensitive to the aminoglycosides tobramycin, gentamicin and amikacin, indicating DDR and SAC activation might rely on translation fidelity, under aminoglycoside stress. Further, we report that the DNA damage induced by aminoglycosides in the base excision repair mutants ogg1Δ and apn1Δ is mediated by reactive oxygen species, which induce the DNA adduct 8-hydroxy deoxyguanosine. Finally, the synergistic effect of tobramycin and the DNA-damaging agent bleomycin to sensitize trm9Δ and the DDR mutants mlh1Δ, rad51Δ, mre11Δ and sgs1Δ at significantly lower concentrations compared with wild-type suggests that cells with tRNA modification dysregulation and DNA repair gene defects can be selectively sensitized using a combination of translation inhibitors and DNA-damaging agents.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Aminoglicosídeos/farmacologia , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo , tRNA Metiltransferases/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Antibacterianos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Reparo do DNA , Dano ao DNA , Tobramicina/farmacologia , RNA de TransferênciaRESUMO
Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier presented by antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocaglates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a eukaryotic translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to the Ophiocordyceps genus and that its eIF4A, a molecular target of rocaglates, harbors an amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered C. orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.
Although plants may seem like passive creatures, they are in fact engaged in a constant battle against the parasitic fungi that attack them. To combat these fungal foes, plants produce small molecules that act like chemical weapons and kill the parasite. However, the fungi sometimes fight back, often by developing enzymes that can break down the deadly chemicals into harmless products. One class of anti-fungal molecules that has drawn great interest is rocaglates, as they show promise as treatments for cancer and COVID-19. Rocaglates are produced by plants in the Aglaia family and work by targeting the fungal molecule eIF4A which is fundamental for synthesizing proteins. Since proteins perform most of the chemistry necessary for life, one might think that rocaglates could ward off any fungus. But Chen et al. discovered there is in fact a species of fungi that can evade this powerful defense mechanism. After seeing this new-found fungal species successfully growing on Aglaia plants, Chen et al. set out to find how it is able to protect itself from rocoglates. Genetic analysis of the fungus revealed that its eIF4A contained a single mutation that 'blocked' rocaglates from interacting with it. Chen et al. confirmed this effect by engineering a second fungal species (which infects cucumber plants) so that its elF4A protein contained the mutation found in the new fungus. Fungi with the mutated eIF4A thrived on cucumber leaves treated with a chemical derived from rocaglates, whereas fungi with the non-mutated version were less successful. These results shed new light on the constant 'arms race' between plants and their fungal parasites, with each side evolving more sophisticated ways to overcome the other's defenses. Chen et al. hope that identifying the new rocaglate-resistant eIF4A mutation will help guide the development and use of any therapies based on rocaglates. Further work investigating how often the mutation occurs in humans will also be important for determining how effective these therapies will be.
Assuntos
Aglaia , Hypocreales , Parasitos , Animais , Substituição de Aminoácidos , MutaçãoRESUMO
Searching for novel compounds with antibiotic activity and understanding their mechanism of action is extremely important. The ribosome is one of the main targets for antibiotics in bacterial cells. Even if the molecule does not suit the clinical application for whatever reasons, an investigation of its mechanism of action can deepen our understanding of the ribosome function. Such data can inform us on how the already used translational inhibitors can be modified. In this study, we demonstrate that 1-(2-oxo-2-((4-phenoxyphenyl).
RESUMO
Over recent years, a growing number of bacterial species have become resistant to clinically relevant antibiotics. Proline-rich antimicrobial peptides (PrAMPs) having a potent antimicrobial activity and a negligible toxicity toward mammalian cells attract attention as new templates for the development of antibiotic drugs. Here, we mined genomes of all living Camelidae species and found a novel family of Bac7-like proline-rich cathelicidins which inhibited bacterial protein synthesis. The N-terminal region of a novel peptide from the alpaca Vicugna pacos named VicBac is responsible for inhibition of bacterial protein synthesis with an IC50 value of 0.5 µM in the E. coli cell-free system whereas the C-terminal region allows the peptide to penetrate bacterial membranes effectively. We also found that the full-length VicBac did not induce bacterial resistance after a two-week selection experiment, unlike the N-terminal truncated analog, which depended on the SbmA transport system. Both pro- and anti-inflammatory action of VicBac and its N-terminal truncated variant on various human cell types was found by multiplex immunoassay. The presence of the C-terminal tail in the natural VicBac does not provide for specific immune-modulatory effects in vitro but enhances the observed impact compared with the truncated analog. The pronounced antibacterial activity of VicBac, along with its moderate adverse effects on mammalian cells, make this molecule a promising scaffold for the development of peptide antibiotics.
RESUMO
Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL-CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome-PTC and peptide exit tunnel.
RESUMO
The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.
Assuntos
Compostos de Bifenilo/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Sítios de Ligação , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Cinética , Simulação de Acoplamento Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteômica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Interfacial inhibitors exert their biological effects through co-association with two macromolecules. The pateamine A (PatA) class of molecules function by stabilizing eukaryotic initiation factor (eIF) 4A RNA helicase onto RNA, resulting in translation initiation inhibition. Here, we present the crystal structure of an eIF4A1:RNA complex bound to an analog of the marine sponge-derived natural product PatA, C5-desmethyl PatA (DMPatA). One end of this small molecule wedges itself between two RNA bases while the other end is cradled by several protein residues. Strikingly, DMPatA interacts with the eIF4A1:RNA complex in an almost identical fashion as rocaglamide A (RocA), despite being completely unrelated from a structural standpoint. The structural data rationalize the ability of PatA analogs to target a wider range of RNA substrates compared to RocA. We define the molecular basis of how DMPatA is able to clamp eIF4A1 onto RNA, imparting potent inhibitory properties to this molecule.
Assuntos
Compostos de Epóxi/química , Fator de Iniciação 4A em Eucariotos/química , Macrolídeos/química , RNA/química , Tiazóis/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação MolecularRESUMO
The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity.
Assuntos
Benzofuranos/farmacologia , RNA Helicases DEAD-box/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Benzofuranos/química , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Inibidores Enzimáticos/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Feminino , Humanos , Masculino , Modelos Moleculares , Conformação MolecularRESUMO
Triple negative breast cancers (TNBC) without BRCA1/2 gene mutation or BRCAness are nowadays the breast malignancies most difficult to treat. Improvement of their treatment, for all phases of the disease, is an important unmet medical need. We analyzed the effect of homoharringtonine (HHT), a natural protein synthesis inhibitor approved for treatment of chronic myeloid leukemia, on four cell lines representing aggressive, BRCA1/2 non-mutated, TNBC genomic categories. We show that HHT inhibits in vitro growth of all cell lines for more than 80%, after 48-72 h exposure to 20-100 ng/mL, the concentrations achievable in human plasma after subcutaneous administration of the drug. HHT, at 100 ng/mL, strongly reduced levels of a major TNBC survival factor, anti-apoptotic protein Mcl-1, after only 2 h of exposure, in all cell lines except MDA-MB-231. Other anti-apoptotic proteins, Bcl-2, survivin and XIAP, were also strongly downregulated. Moreover, in vivo growth of the least sensitive cell line to HHT in vitro, MDA-MB-231, was inhibited for 36.5% in mice, by 1 mg/kg of the drug, given subcutaneously, bi-daily, over 7 days. These results demonstrate marked antineoplastic activity of homoharringtonine in TNBC, making further development of the drug in this disease highly warranted.