Mode of action of the dCK-specific inhibitor (R)-DI-87.

(A) Scheme illustrating the mode of action of the dCK-specific inhibitor (R)-DI-87. S. aureus-derived dAdo and dGuo are pumped into phagocytes via human equilibrative transporter 1 (hENT1). Deoxycytidine kinase (dCK) converts dAdo and dGuo into appropriate deoxyribonucleoside monophosphates thereby triggering an accumulation of apoptosis-stimulating deoxyribonucleoside di- and triphosphates. (R)-DI-87 interferes with this pathway by inhibiting dCK, thus preventing host cell death. (B) Structure of (R)-DI-87.

(R)-DI-87 protects phagocytes from death-effector deoxyribonucleoside-mediated cytotoxicity.

(A-D) Survival rates of human U937 monocyte-like cells (U937) (A, B) or U937-derived macrophages (U937 MФ) (C, D) exposed to dAdo or dGuo in the presence (+) or absence (-) of 1 µM (R)-DI-87. Cells were also exposed to the inhibitor or vehicle only. U937 dCK-/- were included as a control. (E-H) Survival rates of human CD14+ monocytes (E, F) or human monocyte-derived macrophages (HMDMs) (G, H) exposed to dAdo or dGuo in the presence (+) or absence (-) of 1 µM (R)-DI-87. Cells were also exposed to the inhibitor or vehicle only. (I-J) Survival rates of U937 MΦ exposed to rAdsA-derived dAdo (I) or dGuo (J). rAdsA was incubated with dAMP or dGMP and reaction products containing dAdo or dGuo were used to treat phagocytes in the presence (+) or absence (-) of 1 µM (R)-DI-87. Controls lacked rAdsA or deoxyribonucleoside monophosphates, or included reaction buffer only as indicated with + and − symbols. 100 µM (A-B; E-F) or 200 µM (C-D; G-H) of dAdo or dGuo were used to treat the cells. Cell survival rates were analyzed 48 h post-treatment. Data are the mean (± standard deviation [SD]) values from at least three independent determinations. Primary cell experiments include at least three independent donors. Statistically significant differences were analyzed with one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test; ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Selective inhibition of dCK prevents death-effector deoxyribonucleoside-mediated induction of immune cell apoptosis.

(A-D) Analysis of caspase-3 activity in human U937 monocyte-like cells (U937) (A, B) or U937-derived macrophages (U937 MФ) (C, D) exposed to dAdo or dGuo in the presence (+) or absence (-) of 1 µM (R)-DI-87. Cells were also exposed to the inhibitor or vehicle only. U937 dCK-/- were included as a control. Caspase-3 activity was analyzed using a colorimetric assay. (E-F) Immunoblotting of lysates obtained from U937 (E) or U937 MФ (F) exposed to dAdo or dGuo in the presence (+) or absence (-) of 1 µM (R)-DI-87. Controls are indicated (+/– symbols). A specific antibody was used that detects the cleaved (active) form of caspase-3 (α-CASP3). GAPDH was used as a loading control (α-GAPDH). Numbers to the right of blots indicate the migration of molecular weight markers in kilodaltons. (G-H) Analysis of (R)-DI-87-dependent prevention of host cell apoptosis via immunofluorescence microscopy. U937 MФ were exposed to dAdo (G) or dGuo (H) in the presence or absence of 1 µM (R)-DI-87 and stained using FITC-annexin-V/PI. Controls are indicated. White bars depict a length of 100 μm. Representative images are shown. 100 µM (A-B; E) or 200 µM (C-D; F-H) of dAdo or dGuo were used to treat the cells. Apoptosis rates were analyzed 24 h post-treatment. Data are the mean (± standard deviation [SD]) values from three independent determinations. Statistically significant differences were analyzed with one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test; ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

(R)-DI-87 protects against S. aureus invasive disease.

(A, B) Safety assessment of (R)-DI-87 in mice. Cohorts of female C57BL/6 mice were treated with (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol) via oral gavage in 12-hour intervals for 16 days. On day 16, peripheral blood was collected and subjected to a FACS-based immuno-phenotyping approach (A). Panel B indicates the body weight gain during the course of treatment. (C-F) Enumeration of visible surface abscesses and staphylococcal loads in organs of S. aureus-challenged C57BL/6 mice treated with (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol). Mice received (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol) via oral gavage every 12 hours and were challenged with 107 CFU of wild-type S. aureus Newman (WT) or its ΔadsA mutant. Data for female C57BL/6 mice are displayed (n = 8). Bacterial burden was enumerated as log10 CFU per gram of tissue at 5 days post-infection. Horizontal blue bars represent the mean values of visible abscesses per organ (C-D) or indicate the mean CFU count in each cohort (E-F). (G, H) Microscopic images of H&E–stained liver (G) or renal tissues (H) obtained after necropsy of S. aureus-challenged C57BL/6 mice treated with (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol). Mice received (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol) via oral gavage every 12 hours and were challenged with 107 CFU of wild-type S. aureus Newman (WT) or its ΔadsA mutant. Arrows point to immune cell infiltrates (black) or replicating staphylococci (blue). Black bars depict a length of 100 μm. Representative images are shown. Statistically significant differences were analyzed by a two-tailed Student’s t-test (A-B) or with the Kruskal– Wallis test corrected with Dunn’s multiple comparison (C-F). ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

(R)-DI-87-mediated inhibition of dCK enhances macrophage infiltration into staphylococcal abscesses.

(A-P) Immunofluorescence microscopy-based detection of macrophages in liver or renal tissues isolated 5 days after intravenous injection of 107 CFU of wild-type S. aureus Newman (A-H) or its adsA mutant (I-P) into wild type C57BL/6 mice treated with (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol). Mice received (R)-DI-87 (75 mg/kg) or vehicle (40% Captisol) via oral gavage every 12 hours. Magnifications of boxed areas from upper panels are indicated. Thin sections were stained with α-F4/80 antibodies (macrophages; red). Nuclei were labeled with DAPI (blue). White bars shown in the upper panels depict 100 μm length. Representative images are shown. (Q, R) Survival rates of murine bone marrow-derived macrophages (BMDMs) exposed to dAdo (Q) or dGuo (R) in the presence (+) or absence (-) of 1 µM (R)-DI-87. Cells were also exposed to the inhibitor or vehicle only. 200 µM of dAdo or dGuo were used to treat the cells. Cell survival rates were analyzed 48 h post-treatment. Data are the mean (± standard deviation [SD]) values from three independent determinations. Statistically significant differences were analyzed with one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test; ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Proposed model of (R)-DI-87-mediated protection of phagocytes during S. aureus abscess formation.

Diagram illustrating the (R)-DI-87-mediated protection of macrophages during the development of staphylococcal abscesses. While phagocytes get killed by S. aureus-derived death-effector deoxyribonucleosides in vehicle-treated animals, (R)-DI-87 protects macrophages and boosts their infiltration into the deeper cavity of infectious foci thereby enhancing eradication of staphylococci.