Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells.

OBJECTIVES: To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. RESULTS: Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. CONCLUSIONS: The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.

Repurposing Potential of Riluzole as an ITAF Inhibitor in mTOR Therapy Resistant Glioblastoma.

Internal ribosome entry site (IRES)-mediated protein synthesis has been demonstrated to play an important role in resistance to mechanistic target of rapamycin (mTOR) targeted therapies. Previously, we have demonstrated that the IRES trans-acting factor (ITAF), hnRNP A1 is required to promote IRES activity and small molecule inhibitors which bind specifically to this ITAF and curtail IRES activity, leading to mTOR inhibitor sensitivity. Here we report the identification of riluzole (Rilutek®), an FDA-approved drug for amyotrophic lateral sclerosis (ALS), via an in silico docking analysis of FDA-approved compounds, as an inhibitor of hnRNP A1. In a riluzole-bead coupled binding assay and in surface plasmon resonance imaging analyses, riluzole was found to directly bind to hnRNP A1 and inhibited IRES activity via effects on ITAF/RNA-binding. Riluzole also demonstrated synergistic anti-glioblastoma (GBM) affects with mTOR inhibitors in vitro and in GBM xenografts in mice. These data suggest that repurposing riluzole, used in conjunction with mTOR inhibitors, may serve as an effective therapeutic option in glioblastoma.

Synthetic Antibody Binding to a Preorganized RNA Domain of Hepatitis C Virus Internal Ribosome Entry Site Inhibits Translation.

Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). We used these Fabs as crystallization chaperones and determined the high-resolution crystal structures of JIIIabc-HCV2 and -HCV3 complexes at 1.81 Å and 2.75 Å resolution respectively, revealing an antiparallel four-way junction with the IIIa and IIIc subdomains brought together through tertiary interactions. The RNA conformation observed in the structures supports the structural model for this region derived from cryo-EM data for the HCV IRES-40S ribosome complex, suggesting that the tertiary fold of the RNA preorganizes the domain for interactions with the 40S ribosome. Strikingly, both Fabs and the ribosomal protein eS27 not only interact with a common subset of nucleotides within the JIIIabc but also use physiochemically similar sets of protein residues to do so, suggesting that the RNA surface is well-suited for interactions with proteins, perhaps analogous to the "hot spot" concept elaborated for protein-protein interactions. Using a rabbit reticulocyte lysate-based translation assay with a bicistronic reporter construct, we further demonstrated that Fabs HCV2 and HCV3 specifically inhibit the HCV IRES-directed translation, implicating disruption of the JIIIabc-ribosome interaction as a potential therapeutic strategy against HCV.

Quinoxaline derivatives disrupt the base stacking of hepatitis C virus-internal ribosome entry site RNA: reduce translation and replication.

RNA-biased small molecules with a monoquinoxaline core target the L-shaped structure of subdomain IIa of Hepatitis C virus internal ribosome entry site (IRES) RNA in proximity to the Mg2+ binding site. The binding event leads to the destacking of RNA bases, resulting in the inhibition of IRES-mediated translation and HCV RNA replication.

The hidden nature of protein translational control by diphthamide: the secrets under the leather.

The protein translation elongation factor eEF2 undergoes a unique posttranslational modification called diphthamidation. eEF2 is an essential factor in protein translation, and the diphthamide modification has been a famous target of the diphtheria toxin for a long time. On the other hand, the physiological function of this rare modification in vivo remains unknown. Recent studies have suggested that diphthamide has specific functions for the cellular stress response and active proliferation. In this review, we summarize the history and findings of diphthamide obtained to date and discuss the possibility of a specific function for diphthamide in regulating protein translation.

Translational control of the undifferentiated phenotype in ER­positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition.

Using a series of potential biomarkers relevant to mechanisms of protein synthesis, we observed that estrogen receptor (ER)-positive breast tumor cells exist in two distinct yet interconvertible phenotypic states (of roughly equal proportion) which differ in the degree of differentiation and use of IRES-mediated translation. Nascently translated IGF1R in the cytoplasm positively correlated with IRES activity and the undifferentiated phenotype, while epitope accessibility of RACK1, an integral component of the 40S ribosomal subunit, aligned with the more differentiated IRES-off state. When deprived of soluble growth factors, the entire tumor cell population shifted to the undifferentiated phenotype in which IRES-mediated translation was active, facilitating survival under these adverse microenvironmental conditions. However, if IRES-mediated translation was inhibited, the cells instead were forced to transition uniformly to the more differentiated state. Notably, cytoplasmic localization of estrogen receptor α (ERα/ESR1) precisely mirrored the pattern observed with nascent IGF1R, correlating with the undifferentiated IRES-active phenotype. Inhibition of IRES-mediated translation resulted in both a shift in ERα to the nucleus (consistent with differentiation) and a marked decrease in ERα abundance (consistent with the inhibition of ERα synthesis via its IRES). Although breast tumor cells tolerated forced differentiation without extensive loss of their viability, their reproductive capacity was severely compromised. In addition, CDK1 was decreased, connexin 43 eliminated and Myc translation altered as a consequence of IRES inhibition. Isolated or low-density ER-positive breast tumor cells were particularly vulnerable to IRES inhibition, losing the ability to generate viable cohesive colonies, or undergoing massive cell death. Collectively, these results provide further evidence for the integral relationship between IRES-mediated translation and the undifferentiated phenotype and demonstrate how therapeutic manipulation of this specialized mode of protein synthesis may be used to limit the phenotypic plasticity and incapacitate or eliminate these otherwise highly resilient breast tumor cells.

Induction of the p53 Tumor Suppressor in Cancer Cells through Inhibition of Cap-Dependent Translation.

The p53 tumor suppressor plays a critical role in protecting normal cells from malignant transformation. Development of small molecules to reactivate p53 in cancer cells has been an area of intense research. We previously identified an internal ribosomal entry site (IRES) within the 5' untranslated region of p53 mRNA that mediates translation of the p53 mRNA independent of cap-dependent translation. Our results also show that in response to DNA damage, cells switch from cap-dependent translation to cap-independent translation of p53 mRNA. In the present study, we discovered a specific inhibitor of cap-dependent translation, 4EGI-1, that is capable of inducing the accumulation of p53 in cancer cells retaining wild-type p53. Our results show that 4EGI-1 causes an increase in p53 IRES activity, leading to increased translation of p53 mRNA. We also observed that 4EGI-1 induces cancer cell apoptosis in a p53-dependent manner. Furthermore, 4EGI-1 induces p53 in cancer cells without causing DNA double-strand breaks. In conclusion, we discovered a mechanistic link between inhibition of cap-dependent translation and enhanced p53 accumulation. This leads to apoptosis of cancer cells without causing collateral damage to normal cells, thus providing a novel and effective therapeutic strategy for cancer.

Targeting the CACNA1A IRES as a Treatment for Spinocerebellar Ataxia Type 6.

We have discovered that the P/Q-type voltage-gated Ca2+ channel (VGCC) gene, CACNA1A, encodes both the α1A (Cav2.1) subunit and a newly recognized transcription factor, α1ACT, by means of a novel internal ribosomal entry site (IRES) within the α1A C-terminal coding region. α1ACT, when mutated with an expansion of the polyglutamine tract in the C-terminus, gives rise to spinocerebellar ataxia type 6 (SCA6). Because silencing of the entire CACNA1A gene would result in the loss of the essential Cav2.1 channel, the IRES controlling α1ACT expression is an excellent target for selective silencing of α1ACT as a therapeutic intervention for SCA6. We performed a high-throughput screen of FDA-approved small molecules using a dual luciferase reporter system and identified ten hits able to selectively inhibit the IRES. We identified four main candidates that showed selective suppression of α1ACT relative to α1A in HEK cells expressing a native CACNA1A vector. We previously pursued another avenue of molecular intervention through miRNA silencing. We studied three human miRNAs (miRNA-711, -3191-5p, -4786) that would potentially bind to sequences within the CACNA1A IRES region, based on an miRNA prediction program. Only miRNA-3191-5p was found to selectively inhibit the translation of α1ACT in cells. We developed a hyperacute model of SCA6 in mice by injecting a pathogenic form of the IRES-mediated α1ACT (AAV9-α1ACTQ33). Finally, we tested the effectiveness of the miRNA therapy by co-expressing either control miRNA or miRNA-3191-5p and found that miRNA-3191-5p decreased the levels of α1ACTQ33 and prevented the hyperacute disease in mice. These studies provide the proof of principle that a therapy directed at selectively preventing α1ACT expression could be used to treat SCA6.

Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome.

Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome's internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Enteroviruses: Classification, Diseases They Cause, and Approaches to Development of Antiviral Drugs.

The genus Enterovirus combines a portion of small (+)ssRNA-containing viruses and is divided into 10 species of true enteroviruses and three species of rhinoviruses. These viruses are causative agents of the widest spectrum of severe and deadly epidemic diseases of higher vertebrates, including humans. Their ubiquitous distribution and high pathogenicity motivate active search to counteract enterovirus infections. There are no sufficiently effective drugs targeted against enteroviral diseases, thus treatment is reduced to supportive and symptomatic measures. This makes it extremely urgent to develop drugs that directly affect enteroviruses and hinder their development and spread in infected organisms. In this review, we cover the classification of enteroviruses, mention the most common enterovirus infections and their clinical manifestations, and consider the current state of development of anti-enteroviral drugs. One of the most promising targets for such antiviral drugs is the viral Internal Ribosome Entry Site (IRES). The classification of these elements of the viral mRNA translation system is also examined.

Ligand Optimization by Improving Shape Complementarity at a Hepatitis C Virus RNA Target.

Crystal structure analysis revealed key interactions of a 2-amino-benzimidazole viral translation inhibitor that captures an elongated conformation of an RNA switch target in the internal ribosome entry site (IRES) of hepatitis C virus (HCV). Here, we have designed and synthesized quinazoline derivatives with improved shape complementarity at the ligand binding site of the viral RNA target. A spiro-cyclopropyl modification aimed at filling a pocket in the back of the RNA binding site led to a 5-fold increase of ligand affinity while a slightly more voluminous dimethyl substitution at the same position did not improve binding. We demonstrate that precise shape complementarity based solely on hydrophobic interactions contributes significantly to ligand binding even at a hydrophilic RNA target site such as the HCV IRES conformational switch.

Translating Hedgehog in Cancer: Controlling Protein Synthesis.

Developmental Hedgehog (Hh) signaling is found deregulated in a broad spectrum of human malignancies and, thus, is an attractive target for cancer therapy. Currently available Hh inhibitors have shown the rapid occurrence of drug resistance, due to altered signaling in collateral pathways. Emerging observations suggest that Hh signaling regulates protein translation in pathways that depend both on Cap- and IRES-mediated translation. In addition, translational regulators have been shown to modulate Hh function. In this opinion, we describe this novel Hh/translation crosstalk and argue that it plays a relevant role in Hh-mediated tumorigenesis and drug resistance. As such, we suggest that drugs targeting translation might be introduced in novel protocols aimed at treating malignancies driven by aberrant Hh signaling.

Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress.

Protein translation is inhibited by the unfolded protein response (UPR)-induced eIF-2α phosphorylation to protect against endoplasmic reticulum (ER) stress. In addition, we found additional inhibition of protein translation owing to diminished mTORC1 (mammalian target of rapamycin complex1) activity in ER-stressed multiple myeloma (MM) cells. However, c-myc protein levels and myc translation was maintained. To ascertain how c-myc was maintained, we studied myc IRES (internal ribosome entry site) function, which does not require mTORC1 activity. Myc IRES activity was upregulated in MM cells during ER stress induced by thapsigargin, tunicamycin or the myeloma therapeutic bortezomib. IRES activity was dependent on upstream MAPK (mitogen-activated protein kinase) and MNK1 (MAPK-interacting serine/threonine kinase 1) signaling. A screen identified hnRNP A1 (A1) and RPS25 as IRES-binding trans-acting factors required for ER stress-activated activity. A1 associated with RPS25 during ER stress and this was prevented by an MNK inhibitor. In a proof of principle, we identified a compound that prevented binding of A1 to the myc IRES and specifically inhibited myc IRES activity in MM cells. This compound, when used alone, was not cytotoxic nor did it inhibit myc translation or protein expression. However, when combined with ER stress inducers, especially bortezomib, a remarkable synergistic cytotoxicity ensued with associated inhibition of myc translation and expression. These results underscore the potential for targeting A1-mediated myc IRES activity in MM cells during ER stress.

The sirtuin inhibitor sirtinol inhibits hepatitis A virus (HAV) replication by inhibiting HAV internal ribosomal entry site activity.

Epigenetics plays a role in the regulation of gene expression. Epigenetic changes control gene expression at the transcriptional level. Our previous study suggested that the La protein, which is mainly localized in the nucleus, was associated with hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation and HAV replication. The aim of this study was to investigate whether epigenetic compounds have effects on HAV IRES-mediated translation and HAV replication. Sirtinol, a sirtuin inhibitor, inhibited HAV IRES-mediated translation in COS7-HAV-IRES cells. Treatment with 10 µM sirtinol resulted in a significant reduction in the intracellular RNA levels of HAV HA11-1299 genotype IIIA in Huh7 cells. Epigenetic treatment with a sirtuin inhibitor may represent a new treatment option for HAV infection. In conclusion, epigenetic control was involved in HAV IRES-dependent translation and HAV replication. Special attention should also be paid to underlying viral diseases in the clinical use of epigenetic treatments for malignancies.

Apigenin restricts FMDV infection and inhibits viral IRES driven translational activity.

Foot-and-mouth disease (FMD) is a highly contagious disease of domestic and wild ruminants that is caused by FMD virus (FMDV). FMD outbreaks have occurred in livestock-containing regions worldwide. Apigenin, which is a flavonoid naturally existing in plant, possesses various pharmacological effects, including anti-inflammatory, anticancer, antioxidant and antiviral activities. Results show that apigenin can inhibit FMDV-mediated cytopathogenic effect and FMDV replication in vitro. Further studies demonstrate the following: (i) apigenin inhibits FMDV infection at the viral post-entry stage; (ii) apigenin does not exhibit direct extracellular virucidal activity; and (iii) apigenin interferes with the translational activity of FMDV driven by internal ribosome entry site. Studies on applying apigein in vivo are required for drug development and further identification of potential drug targets against FDMV infection.

Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5'-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.