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

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

4-Hydroxybenzaldehyde (28 mg, 0.23 mmol) was dissolved in anhydrous acetonitrile (2 mL), added to cesium carbonate (112 mg, 0.34 mmol) and stirred for 15 min at room temperature. A solution of "acetylene-TEG-I" (117 mg, 0.34 mmol) in anhydrous MeCN (2 mL) was added and the stirring reaction mixture was reduced to 1 mL under nitrogen. After 72 h it was reduced to dryness under nitrogen. The residue was taken up in chloroform and loaded onto a silica column equilibrated in 99:1 chloroform:methanol. The column was developed with a gradient of 1-5% methanol:chloroform and 20 fractions were collected. Fractions 7-12 were combined (TLC) and reduced to dryness in vacuo yielding acetylene-TEG-BA as a light-yellow oil (74.2 mg, 96.4%). 1  Biotin-TEG-triazole-TEG-BA. Copper(II) sulfate (0.1 mM; 0.25 mL, 23 µmol) was added to a solution of sodium ascorbate (9.1 mg, 46 µmol) in tert-butanol (0.25 mL) and stirred until the initially forming brown precipitate turned to a yellow solution (5 min). To this, a solution of Biotin-TEG-N 3 (34 mg, 76.5 µmol) and acetylene-TEG-BA (30.8 mg, 91.8 µmol) in tert-butanol:water (1:1, 1 mL) was added and stirred for 18 h. The reaction mixture was reduced under nitrogen and freeze-dried. The residue was suspended in 98:2 chloroform:methanol and loaded onto a silica column equilibrated in 99:1 chloroform:methanol. A   To this, NaBH(OAc) 3  2D-NMR data (HSQC, HMBC, COSY, ROESY and NOESY) acquired for B-DAP showed that it was approximately a 1:2 mixture of B-DAP and of an oxidized analogue incorporating biotin sulfoxide (B Ox -DAP). This is manifested as two subsets of urea NH signals (δ H 6.78 / 6.69 and 6.42 / 6.37) in the HMBC and COSY data ( Figure S2) [2]. MS studies (below) provided further evidence for the presence of B-DAP and B Ox -DAP.
In Fragmentation was performed via alternating collision induced detection (CID) and electron transfer detection (ETD). In MS 2 of the above parent ion, the major two fragments were identified as the biotinylated linker including the amine group of ornithine and the potassium salt of daptomycin lacking the ornithine amino group ( Figure S3, panel C). MS 3 studies of the daptomycin fragment revealed two primary fragmentation pathways of nor-daptomycin ( Figures S4 and S5), i.e. pathways 2A and 2B. The starting point for both pathways was the potassium adduct of nor-daptomycin. Pathway 2A started with a ring opening between Nor6 and Asp7 and included an immediate loss of water. In parallel, pathway 2B was observed, which showed a ring-opening of nor-daptomycin at the lactone between Thr4 and Kyn13. Further fragmentation patterns matched the amino acid sequences for the ring opened initial fragment. Due to the initial loss of the TEG-biotin with an amino group and the consequent fragmentation pattern showing that the rest of the molecule was intact, it was concluded that reductive amination had occurred on the ornithine side chain as outlined in Figure S4.
For simplicity reasons, the mixture of B-DAP and B Ox -DAP are further referred to as B-DAP. Purity test of B-DAP. The purity of B-DAP was verified by reinjecting an aliquot (30 µL; 0.21 µM in 20% acetonitrile:5 mM ammonium phosphate buffer, pH =3.2) onto an analytical C18 RP-HPLC column (1 mL/min, 20-70% acetonitrile:5mM ammonium phosphate buffer over 30 min). The trace contained exclusively one peak, which eluted after 21.8 min and showed absorbance maxima at 224 nm, 259 nm and 369 nm ( Figure S6, top panel).

Stability test of B-DAP.
A solution of B-DAP in PBS was incubated at room temperature and aliquots analyzed by RP-HPLC over 12 h on an analytical C18 RP-HPLC column (1 mL/min, 25-80% acetonitrile:5 mM ammonium phosphate buffer (pH =3.2) over 40 min; Figure S6, bottom panel). Integration ( Figure S6, insert) indicated that hydrolysis of B-DAP occurs very slowly (1% over 12 h), and hence B-DAP was regarded stable enough for the biopanning experiments (~ 6 h). PBS was used as a negative control.
Antibiotic activity of B-DAP. Biosafety approval was obtained from the Macquarie University Biosafety Committee (approval number 5201000870). The strains of bacteria used was Staphylococcus aureus ATCC 9144 (obtained from the CDS Reference Laboratory, Department of Microbiology, The Prince of Wales Hospital, NSW, Australia). Glycerol stock cultures (20% v/v glycerol) of the strain were kept at -80 °C. Initial cultures were prepared by streaking a small quantity of the frozen stock onto an LB agar plate and incubation at 37 °C for 16 h. The plates were stored at 4°C and used to inoculate subsequent cultures for up to one month. Overnight cultures were generally prepared by inoculating the according medium with a single bacterial colony from the agar plates, and subsequent incubation at 37 7 °C for 16 h with shaking (100-150 rpm). The optical density at 600 nm (OD 600 ) was adjusted to 0.08-0.1 to produce an inoculum density of 1.0 × 10 8 cfu/mL (OD). For the turbidity microdilution assay, the bacterial strain was grown overnight in Mueller Hinton broth (MHB) (15 mL). The assay was performed in sterile, clear flat-bottom-96-well microtiter plates. The arrangement of samples and controls was outlined according to Appendino [3]. DAP, B-DAP or ampicillin (5.0 mg) were dissolved in DMSO (1 mL) and the final volume was made up to 5.0 mL with distilled water. Using a 96 well microtiter plate, MHB was dispensed into wells 1-11 (125 µL each) for each row, the antibiotic solution was added to well 1 (125 µL; in different rows for each antibiotic/replicate) and mixed thoroughly, after which 125 µL was taken out and dispensed to the next well (i.e. well 2). This process of two-fold serial dilution was carried out until well 10, and skipping well 11, the final volume was dispensed into well 12. Again, 125 µL each of the bacterial inoculum was dispensed into wells 1 to 11 leaving well 12 free of inoculum. Well 11 was free of the test or control compound, thus this acted as a positive growth control. Similarly, well 12 served as sterile control of the assay. 5% DMSO was also included as a negative control. The optical density at 600 nm (OD 600 ) was measured and the plate was incubated at 37 °C for 18 h. After incubation, the OD 600 was determined again and the pre-incubation values subtracted. The resulting optical densities were converted to %(growth), plotted against molar concentration of the compounds and analyzed in GraphPad Prism to yield LD 50 values (Fig. S8).