Volume 23, Issue 8, Pages 1002-1013 (August 2016) Structure-Based Identification of a Potent Inhibitor Targeting Stp1-Mediated Virulence Regulation in Staphylococcus aureus  Weihao Zheng, Xiaodan Cai, Mingsheng Xie, Yujie Liang, Tao Wang, Zigang Li  Cell Chemical Biology  Volume 23, Issue 8, Pages 1002-1013 (August 2016) DOI: 10.1016/j.chembiol.2016.06.014 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 1002-1013DOI: (10. 1016/j. chembiol Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Structure and the Fourth Metal Ion of Stp1 (A) View of the edge of the β-sandwich structure of Stp1. Metal ions (M1–M4) are colored in cyan. (B) View of the catalytic site from above, looking down on the β-sandwich structure of Stp1 (perpendicular to Figure 1A). (C) Stereo view of the fourth metal ion (M4) and selected amino acid residues. (D) Phosphatase activities of Stp1 variants. Stk1 (2 μM) was incubated with ATP in 20 μL of buffer P at 37°C for 30 min, followed by addition of Stp1 (0.5 μM) for an additional 10-min incubation. All samples were analyzed by western blot. Thr-Pi was detected using an anti-phosphothreonine antibody. To ensure loading quality, we subjected similar amounts of Stk1 to electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie R250. Relative phosphointensity was calculated by Image Lab software (Bio-Rad). The data represent the mean ± SEM of three independent experiments. See also Figures S1–S3; Tables S1 and S2. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Identification and Mechanistic Studies of Stp1 Inhibitor ATA (A) Structure and activity relationship studies of ATA using the pNPP phosphatase assay. Stp1 (0.1 μM) was incubated with inhibitors at room temperature for 10 min, pNPP (0.75 mM) was added, and the reaction was incubated at room temperature for 20 min, after which the absorbance at 405 nm was recorded. Data represent the mean ± SEM, n = 3 independent experiments. (B) ATA prevented Stp1 dephosphorylating Stk1 while there was no effect on the autophosphorylation activity of Stk1. Stk1 (2 μM) was incubated with ATP in 20 μL of buffer P lacking ATA at 37°C for 30 min and different amounts of ATA were added, followed by addition of Stp1 (0.1 μM) for an additional 10-min incubation. All samples were analyzed by western blot. Thr-Pi was detected using an anti-phosphothreonine antibody. To ensure loading quality, we subjected similar amounts of Stk1 to electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie R250. (C) Direct binding of ATA to Stp1 was tested with surface plasmon resonance, and the dissociation constant (KD = 2.12 μM) was calculated. See also Figure S4. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Binding of ATA to Stp1 (A) Molecular docking experiment indicated that ATA (green) lies in a clover-like pocket located above the dinuclear metal center and near the flap domain. Residues that interacted with ATA are colored blue. (B) Stereo view of the binding site of ATA and selective amino acid residues. (C) Amino acid substitutions studies via pNPP phosphatase assay. Proteins (0.1 μM) were incubated with ATA (0, 1 μM, and 5 μM) at room temperature for 10 min, then pNPP (0.75 mM) was added and the reaction was incubated at room temperature for 20 min, after which the absorbance at 405 nm was recorded. (D) Both the N162A and D198A substitutions reduced the stabilized effect (ΔTm) of ATA on the enzymes via thermal-shift experiment. All data represent the mean ± SEM, n = 3 independent experiments. *p < 0.01, **p < 0.001 by two-tailed Student's t test. See also Figure S4 and Table S2. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Potency and Selectivity of ATA (A) ATA demonstrated enhanced inhibition in comparison with canonical phosphatase inhibitors via pNPP phosphatase assay. Data represent the mean ± SEM, n = 3 independent experiments. (B) ATA showed superior inhibition effect on Stp1 dephosphorylating Stk1. All inhibitors had negligible effects on the autophosphorylation activity of Stk1. Stk1 (2 μM) was incubated with ATP in 20 μL of buffer P lacking inhibitors at 37°C for 30 min and 100 μM inhibitors was added, followed by addition of Stp1 (0.1 μM) for an additional 10-min incubation. All samples were analyzed by western blot. Thr-Pi was detected using an anti-phosphothreonine antibody. To ensure loading quality, we subjected similar amounts of Stk1 to electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie R250. (C) ATA inhibited Stp1 effectively, whereas only mild inhibition was observed using PP2Cα and MspP. Data were obtained via pNPP phosphatase assay and represent the mean ± SEM, n = 3 independent experiments. See also Table S3. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 ATA Inhibits stp1-Regulated Gene Transcription and Secreted Virulence Factor Production (A) Venn diagram showing the number of differentially expressed genes for the ATA-treated group (WT + ATA versus WT) and Δstp1 group (Δstp1 versus WT). (B) Cluster enrichment analysis indicates that 200 differentially expressed genes were shared between the ATA-treated group and Δstp1 group. Genes with |log2 ratio| ≥ 0.5 and a false discovery rate of ≤0.05 were considered as differentially expressed genes. (C) Selected differentially expressed genes were confirmed by qRT-PCR. Data represent the mean ± SEM, n = 3 independent experiments. (D) Western blot analysis of Hla production as described in Supplemental Experimental Procedures. (E) Hemolysis on the sheep blood agar. The strains with or without ATA treatment were spotted on a 5% (v/v) sheep blood agar plate. Zones of clearance indicate hemolysis. (F) Triton X-100-induced autolysis assay under static conditions. The autolysis of mid-exponential-phase cultures was determined at 37°C in the presence of 0.05% Triton X-100 by measuring decreases in optical densities upon exposure to the detergent. The asterisks indicate statistical significance of the autolysis rate of WT + ATA versus WT, Δstp1/pYJ335-stp1+ATA versus Δstp1/pYJ335-stp1, and Δstp1 versus WT by two-tailed Student's t test (**p < 0.001). Data represent the mean ± SEM, n = 3 independent experiments. See also Figure S5 and Data S1. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 ATA Interacts with Stp1 and Enhances SarA/MgrA Phosphorylation in S. aureus (A and B) Western blot image (A) and the cellular thermal-shift assay curves (B) show that ATA (100 μM) increased the thermal stability of Stp1 protein in the Δstp1/pYJ335-stp1 strain cells. Data represent the mean ± SEM, n = 3 independent experiments. (C–E) ATA enhanced SarA (C) and MgrA (D) phosphorylation in S. aureus. (E) ATA had a negligible effect on SarA phosphorylation in the stp1-deficient strain. His6-SarA and His6-MgrA were enriched with Ni-NTA beads from whole-cell extracts of ΔsarA/pYJ335-sarA and Δstp1-sarA/pYJ335-sarA, and ΔmgrA/pYJ335-mgrA, respectively, after treatment with various amounts of ATA. To ensure loading quality, we subjected similar amounts of SarA or MgrA to electrophoresis on a 12% SDS-PAGE gel and stained with Coomassie R250. Recombinant SarA or MgrA expressed by E. coli served as a negative control. Pi was detected using an anti-phosphothreonine antibody. See also Figure S6. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 ATA Attenuates Staphylococcal Virulence in a Murine Abscess Formation Model (A) BALB/c mice (n = 10) received mock (control) or ATA (15 mg/kg/day) treatment via intraperitoneal injection from day 0 to day 4. Four hours after the first injection, animals were infected via intravenous inoculation of 1 × 107 CFUs of S. aureus Newman strain. After 5 days, livers and kidneys were harvested and bacterial CFUs in the organs were measured by the serial dilution method. In the graph, each dot represents a mouse. The statistical difference was determined by a two-tailed Student's t test. (B) Representative micrographs of kidney and liver histopathological sections from mice stained by H&E. The control liver and kidneys showed no pathological changes. Mice infected with S. aureus Newman strain had intense infiltration of inflammatory cells in kidneys (white arrow). Infected mice treated with ATA (15 mg/kg/day) showed no significant changes, displaying healthy cells. Mouse livers treated with ATA (15 mg/kg/day) exhibited nearly normal structures of hepatocytes, while untreated infected mice had vacuolated cytoplasm nuclei and karyopyknosis of hepatocytes. Asterisk indicates the pelvis. Scale bars, 500 μm (magnification 200×). See also Figure S7. Cell Chemical Biology 2016 23, 1002-1013DOI: (10.1016/j.chembiol.2016.06.014) Copyright © 2016 Elsevier Ltd Terms and Conditions