Establishment of an imaging-based screening pipeline for the identification of human ribosome biogenesis inhibitors Article Swipe
Claudia Gafko
,
Réka Hollandi
,
Kerstin Dörner
,
Matteo Rosellini
,
Ivo Zemp
,
Péter Horváth
,
Ulrike Kutay
·
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1186/s12915-025-02425-2
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1186/s12915-025-02425-2
Background Ribosomes are huge ribonucleoprotein particles that mediate protein synthesis in all organisms. The synthesis of ribosomes is a complex process that involves hundreds of supporting factors in mammalian cells, including proto-oncogenes and tumor suppressors. Dysregulation of ribosome biogenesis can contribute to tumorigenesis, and the increased production of ribosomes in cancer cells is known to promote proliferative cell growth. Therefore, ribosome biogenesis represents an attractive vulnerability of cancer cells that ought to be exploited for the development of anti-cancer drugs. Despite the large number of trans-acting factors promoting ribosome assembly including potentially druggable enzymes, only few chemical inhibitors that act on ribosome biogenesis, especially downstream of pre-rRNA transcription, have been identified to date. Results To enable large-scale screens for chemical compounds that interfere with ribosome biogenesis, we have established a pipeline to perform single-cell, imaging-based screening campaigns using four different readouts, including fluorescent ribosomal protein reporters (RPS2-YFP, RPL29-GFP) and immunofluorescence analyses of the ribosome biogenesis factor ENP1(BYSL), in HeLa cells, a human cancer line. We have assessed the robustness of our high-content screening approach by performing a pilot screen using a library comprising more than 1000 FDA-approved drugs with known targets in other pathways. This pilot screen obtained excellent quality scores and identified ten compounds as hits. These hit compounds likely affect ribosome synthesis indirectly, the majority by inducing DNA damage or by inhibiting the proteasome. We therefore used the identified compounds to establish appropriate counter assays for DNA damage and proteasome inhibition, to exclude common indirect effects in the downstream analysis of such screening campaigns. Conclusions The established screening pipelines provide a robust, efficient, and sensitive experimental framework to identify chemical compounds that impair ribosome synthesis. The combination of readouts allows to distinguish effects on pre-rRNA synthesis from downstream effects on ribosome assembly. Established counter assays on DNA damage and protein degradation enable to exclude effects on these pathways, which commonly interfere with ribosome synthesis indirectly. The developed assays are easily scalable to screen libraries of higher complexity in the future.
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- article
- Language
- en
- Landing Page
- https://doi.org/10.1186/s12915-025-02425-2
- https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-025-02425-2
- OA Status
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- References
- 86
- OpenAlex ID
- https://openalex.org/W4415381293
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Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4415381293Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1186/s12915-025-02425-2Digital Object Identifier
- Title
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Establishment of an imaging-based screening pipeline for the identification of human ribosome biogenesis inhibitorsWork title
- Type
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articleOpenAlex work type
- Language
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enPrimary language
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2025Year of publication
- Publication date
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2025-10-21Full publication date if available
- Authors
-
Claudia Gafko, Réka Hollandi, Kerstin Dörner, Matteo Rosellini, Ivo Zemp, Péter Horváth, Ulrike KutayList of authors in order
- Landing page
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https://doi.org/10.1186/s12915-025-02425-2Publisher landing page
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https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-025-02425-2Direct link to full text PDF
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goldOpen access status per OpenAlex
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https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-025-02425-2Direct OA link when available
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0Total citation count in OpenAlex
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86Number of works referenced by this work
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