Abstract
Background
Daptomycin is a recently introduced, last-resort antibiotic that displays a unique mode of action against Gram-positive bacteria that is not fully understood. Several bacterial targets have been proposed but no human binding partner is known.
Methods
In the present study we tested daptomycin in cell viability and proliferation assays against six human cell lines, describe the synthesis of biotinylated and fluorescently labeled analogues of daptomycin. Biotinylated daptomycin was used as bait to isolate the human binding partner by the application of reverse chemical proteomics using T7 phage display of five human tumor cDNA libraries. The interaction between the rescued protein and daptomycin was validated via siRNA knockdown, DARTS assay and immunocytochemistry.
Results
We have found that daptomycin possesses selective growth inhibition of some cancer cell lines, especially MCF7. The unbiased interrogation of human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of daptomycin; ribosomal protein S19. Using a drug affinity responsive target stability (DARTS) assay in vitro, we show that daptomycin stabilizes RPS19 toward pronase. Fluorescently labeled daptomycin stained specific structures in HeLa cells and co-localized with a RPS19 antibody.
Conclusion
This study provides, for the first time, a human protein target of daptomycin and identifies RPS19 as a possible anticancer drug target for the development of new pharmacological applications and research.
Daptomycin is a recently introduced, last-resort antibiotic that displays a unique mode of action against Gram-positive bacteria that is not fully understood. Several bacterial targets have been proposed but no human binding partner is known.
Methods
In the present study we tested daptomycin in cell viability and proliferation assays against six human cell lines, describe the synthesis of biotinylated and fluorescently labeled analogues of daptomycin. Biotinylated daptomycin was used as bait to isolate the human binding partner by the application of reverse chemical proteomics using T7 phage display of five human tumor cDNA libraries. The interaction between the rescued protein and daptomycin was validated via siRNA knockdown, DARTS assay and immunocytochemistry.
Results
We have found that daptomycin possesses selective growth inhibition of some cancer cell lines, especially MCF7. The unbiased interrogation of human cDNA libraries, displayed on bacteriophage T7, revealed a single human target of daptomycin; ribosomal protein S19. Using a drug affinity responsive target stability (DARTS) assay in vitro, we show that daptomycin stabilizes RPS19 toward pronase. Fluorescently labeled daptomycin stained specific structures in HeLa cells and co-localized with a RPS19 antibody.
Conclusion
This study provides, for the first time, a human protein target of daptomycin and identifies RPS19 as a possible anticancer drug target for the development of new pharmacological applications and research.
| Original language | English |
|---|---|
| Article number | 16 |
| Pages (from-to) | 1-15 |
| Number of pages | 15 |
| Journal | Proteome Science |
| Volume | 15 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1 Jul 2017 |
Bibliographical note
Copyright the Author(s) 2017. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.Keywords
- daptomycin
- reverse chemical proteomics
- phage display
- DARTS