Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

Michael Gotsbacher, Sungmin Cho, Ho Jeong Kwon, Peter Karuso

Research output: Contribution to journalArticleResearchpeer-review

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.
LanguageEnglish
Article number16
Pages1-15
Number of pages15
JournalProteome Science
Volume15
Issue number1
DOIs
Publication statusPublished - 1 Jul 2017

Fingerprint

Daptomycin
Anti-Bacterial Agents
Bacteriophage T7
Assays
Bacteriophages
Cells
Gene Library
Pharmaceutical Preparations
ribosomal protein S19
Cell Line
Pronase
Gram-Positive Bacteria
HeLa Cells
Proteomics
Small Interfering RNA
Tumors
Neoplasms
Cell Survival
Bacteria
Proteins

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

Cite this

Gotsbacher, Michael ; Cho, Sungmin ; Kwon, Ho Jeong ; Karuso, Peter. / Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans. In: Proteome Science. 2017 ; Vol. 15, No. 1. pp. 1-15.
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Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans. / Gotsbacher, Michael; Cho, Sungmin; Kwon, Ho Jeong; Karuso, Peter.

In: Proteome Science, Vol. 15, No. 1, 16, 01.07.2017, p. 1-15.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Cho, Sungmin

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N2 - BackgroundDaptomycin 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.MethodsIn 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.ResultsWe 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.ConclusionThis 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.

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