Novel fluorescent cycloheximide derivatives for the imaging of protein synthesis

Paoletti, Francesca; Ainger, Kevin; Donati, Ivan; Scardigli, Raffaella; Vetere, Amedeo; Cattaneo, Antonino; Campa, Cristiana

doi:10.1016/j.bbrc.2010.04.075

Cited by 7 publications

(3 citation statements)

References 13 publications

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“…During retesting eight compounds were confirmed as effective in reducing HBV DNA and their names as well as reported functions and mechanisms are shown in the Table 1. One of these identified hits, cycloheximide, is a known inhibitor of protein synthesis and inducer of cell stress (Paoletti et al, 2010). Because of its known mechanism, the reduction of HBV DNA in cycloheximide treated cells most likely was caused by non-selective cellular toxicity and thus was not pursued further.…”

Section: Resultsmentioning

confidence: 99%

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system

et al. 2013

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There are now 7 nucleoside/tide analogues, along with interferon-α, that are approved by the FDA for the management of chronic hepatitis B virus (HBV) infection, a disease affecting hundreds of millions of people worldwide. These medications, however, are limited in usefulness, and significant side effects and the emergence of viral escape mutants make the development of novel and updated therapeutics a pressing need in the treatment of HBV. With this in mind, a library containing 2,000 compounds already known to be safe in both humans and mice with known mechanisms of action in mammalian cells were tested for the possibility of either antiviral activity against HBV or selective toxicity in HBV producing cell lines. A modified real-time immune-absorbance-polymerase chain reaction (IA-PCR) assay was developed for this screen, utilizing cells that produce and secrete intact HBV virions. In this procedure, viral particles are first captured by an anti-HBs antibody immobilized on a plate. The viral load is subsequently assessed by real-time PCR directly on captured particles. Using this assay, eight compounds were shown to consistently reduce the amount of secreted HBV viral particles in the culture medium under conditions that had no detectable impact on cell viability. Two compounds, proparacaine and chlorophyllide, were shown to reduce HBV levels 4- to 6-fold with an IC50 of 1 and 1.5μM respectively, and were selected for further study. The identification of these compounds as promising antiviral drug candidates against HBV, despite a lack of previous recognition of HBV antiviral activity, supports the validity and utility of testing known compounds for “off- pathogen target” activity against HBV, and also validates this IA-PCR assay as an important tool for the detection of anti-viral activity against enveloped viruses.

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Section: Resultsmentioning

confidence: 99%

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system

et al. 2013

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“…This currently include the use of fluorescent derivatives of cycloheximide to identify a specific site of protein synthesis (25) transfection of genetically engineered vectors for the expression of tagged mRNAs to visualize growing nascent peptide at sites of specific mRNA localization (26); expression of tagged translation initiation factors that serve as FRET pairs to specifically monitor translation initiation (24); and in situ spectral monitoring using the ratio between the peak areas of RNA and proteins as a measure of mRNA translation (28). The results presented in the current study demonstrate the ability of FtTM to quantitatively measure the process of translation and delineate the intracellular topography of protein synthesis activity under normal physiological conditions, in single cells and at subcellular resolution.…”

Section: Discussionmentioning

confidence: 99%

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

Barhoom

Kaur

Cooperman

et al. 2011

Nucleic Acids Research

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We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

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“…The scientific interest for CHX has, however, declined because of its toxicity level, which is too high for the application in humans and because no CHX analogues showed any improvement in activity or toxicity [127,128]. Recently, cycloheximide was derivatized as a fluorescent probe for protein synthesis imaging [129], and also tested as inhibitor of FKBP12 [130] or as an antiviral [131]. These studies have shown that most of the molecular features are essential to maintain its activity ( Figure 6).…”

Section: Cryo-em-based Drug Design Of Cycloheximidementioning

confidence: 99%

Targeting the Human 80S Ribosome in Cancer: From Structure to Function and Drug Design for Innovative Adjuvant Therapeutic Strategies

Gilles

Fréchin

Natchiar

et al. 2020

Cells

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The human 80S ribosome is the cellular nucleoprotein nanomachine in charge of protein synthesis that is profoundly affected during cancer transformation by oncogenic proteins and provides cancerous proliferating cells with proteins and therefore biomass. Indeed, cancer is associated with an increase in ribosome biogenesis and mutations in several ribosomal proteins genes are found in ribosomopathies, which are congenital diseases that display an elevated risk of cancer. Ribosomes and their biogenesis therefore represent attractive anti-cancer targets and several strategies are being developed to identify efficient and specific drugs. Homoharringtonine (HHT) is the only direct ribosome inhibitor currently used in clinics for cancer treatments, although many classical chemotherapeutic drugs also appear to impact on protein synthesis. Here we review the role of the human ribosome as a medical target in cancer, and how functional and structural analysis combined with chemical synthesis of new inhibitors can synergize. The possible existence of oncoribosomes is also discussed. The emerging idea is that targeting the human ribosome could not only allow the interference with cancer cell addiction towards protein synthesis and possibly induce their death but may also be highly valuable to decrease the levels of oncogenic proteins that display a high turnover rate (MYC, MCL1). Cryo-electron microscopy (cryo-EM) is an advanced method that allows the visualization of human ribosome complexes with factors and bound inhibitors to improve our understanding of their functioning mechanisms mode. Cryo-EM structures could greatly assist the foundation phase of a novel drug-design strategy. One goal would be to identify new specific and active molecules targeting the ribosome in cancer such as derivatives of cycloheximide, a well-known ribosome inhibitor.

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Novel fluorescent cycloheximide derivatives for the imaging of protein synthesis

Cited by 7 publications

References 13 publications

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

Targeting the Human 80S Ribosome in Cancer: From Structure to Function and Drug Design for Innovative Adjuvant Therapeutic Strategies

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