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. (K, L) Survival of vehicle- (-) or (R)-DI-87-exposed (+) U937 MΦ after treatment with culture medium (RPMI) that had been conditioned by incubation with either wild-type S. aureus Newman (WT) or its adsA mutant (ΔadsA) in the presence or absence of dAMP (K) or dGMP (L) as indicated with + and – symbols. Controls are indicated. 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 (A-J) or 24 h (K, L) 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 by two-way (A-D) or one-way (E-L) analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test; ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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 can also detect 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 blots and 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 by two-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test; ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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

(A-D) 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 23 days. On day 16, peripheral blood was collected and subjected to a FACS-based immuno-phenotyping approach (A). Subsequent panels indicate the cellularity of spleen (B) and thymus (C) tissues along with the body weight of mice (D) on day 23. (E) Analysis of deoxycytidine (dC) content in mouse plasma following continuous dCK inhibitor treatment on day 23. (F-I) 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 adsA mutant (ΔadsA). 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 (F-G) or indicate the mean CFU count in each cohort (H-I). (J) Microscopic images of H&E–stained liver or renal tissues 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 adsA mutant (ΔadsA). 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-E) or with the Kruskal–Wallis test corrected with Dunn’s multiple comparison (F-I). ns, not significant (P ≥ 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

(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 (WT) (A-H) or its adsA mutant (ΔadsA) (I-P) into female 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. White arrows point at the periphery of infectious foci (dashed lines). Magnifications of lesions from upper panels are indicated. Asterisk symbols define the region enlarged in the magnification counterpart images. 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 female mice-derived 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 by one-way analysis of variance (ANOVA) followed by 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.