Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humansReport as inadecuate




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Proteome Science

, 15:16

Chemical Proteomics

Abstract

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.

KeywordsDaptomycin Reverse chemical proteomics Phage display DARTS AbbreviationsBAbenzylaldehyde

B-DAPbiotinylated daptomycin

B-PROPbiotinylated propylamine

CRYcryptopleurine

DAPdaptomycin

DARTSdrug affinity responsive target stability

F-DAPfluorescent daptomycin

RPS19ribosomal protein S19

RPS19AbAntibody against ribosomal protein S19

siRPS19siRNA against RPS19

TEGtriethyleneglycol

Electronic supplementary materialThe online version of this article doi:10.1186-s12953-017-0124-2 contains supplementary material, which is available to authorized users.

Michael P. Gotsbacher and Sungmin Cho are co-first authors.

Ho Jeong Kwon and Peter Karuso are co-corresponding authors.

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Author: Michael P. Gotsbacher - Sungmin Cho - Ho Jeong Kwon - Peter Karuso

Source: https://link.springer.com/







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