Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032 accelerates wound healing in both mouse and rabbit models. A ) The structure of ZLY032, the wound generation and photo acquisition model diagram, wound size analysis flowchart. B) Left panel: representative photographs showing the time‐dependent closure of wounds in male mice and the wound healing‐promoting effect of ZLY032. PDGF was acted as positive control. Middle and right panel: the wound area and wound closure rate of each male mice at varying time points. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL (0.1% DMSO); ## p < 0.01 versus PDGF; n = 14 for each group. Scale bar: 2 mm. (Mean ± SD; two‐ way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) C) Left panel: representative photographs showing the time‐dependent closure of wounds in rabbits. PDGF was acted as positive control. Middle and right panel: the wound area and wound closure rate of each rabbit at varying time points. * p < 0.05, *** p < 0.001 versus CTRL; # p < 0.05 versus PDGF; n = 6 for each group. (Mean ± SD; two‐ way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) D–F) The blood flow at the wound site was detected by Doppler blood flow detector on day 8 after the administration of ZLY032 and PDGF. * p < 0.05, *** p < 0.001 versus CTRL, n = 5. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) G) Representative examples of Masson staining showing the effects of ZLY032 in microvessels and distance between epithelial tips (as indicated by the shortened distance between the black arrows) during the healing process (days 8). * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL; # p < 0.05 versus PDGF; n = 3. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Positive Control, Staining

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032 promotes angiogenesis and exerts anti‐inflammatory effects. A) Effect of ZLY032 (5 µ m ) combined with single or dual PPARδ/FFA1 knockdown on HUVECs activity as assessed by CCK‐8 assay. *** p < 0.001 versus siNC‐h, ### p < 0.001 versus siNC‐h +ZLY032 (5 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) B) Effect of ZLY032 (5 µ m ) combined with single or dual PPARδ/FFA1 knockdown on HUVECs migratory capacity as assessed by scratch assay. *** p < 0.001 versus siNC‐h; ### p < 0.001 versus siNC‐h+ZLY032 (5 µ m ); n = 5 for each group. (Mean ± SD; two‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) C) Effect of ZLY032 (5 µ m ) combined with single or dual PPARδ/FFA1 knockdown on HUVECs tube formation capacity as assessed by tube formation assay. *** p < 0.001 versus siNC‐h; ### p < 0.001 versus siNC‐h+ZLY032(5 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) D–G) qRT‐PCR to detect the effects on the expression of CD206, Arg1, Tgfβ1, and Fizz1 in the ZLY032 (5 µ m ) combined with single or dual PPARδ/FFA1 knockdown in RAW264.7 cells. *** p < 0.001 versus siNC‐m; ### p < 0.001 versus siNC‐m+ZLY032 (5 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) H–K) qRT‐PCR was used to detect the effects of ZLY032 (5 µ m ) in combination with single or combined knockdown of PPARδ and FFA1 on the expression of iNos, Tnfα, IL‐10, and Vegfa in LPS stimulated RAW264.7 cells. *** p < 0.001 versus siNC‐m; ### p < 0.001 versus LPS+siNC‐m; && p < 0.01, &&& p < 0.001 versus LPS+siNC‐m+ZLY032 (5 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Knockdown, Activity Assay, CCK-8 Assay, Wound Healing Assay, Tube Formation Assay, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032 promotes angiogenesis and exerts anti‐inflammatory effects by activating PPARδ and FFA1. A) The wound generation and photo acquisition model diagram and wound size analysis flowchart. B) Up panel: representative photographs showing the time‐dependent closure of wounds in male mice and the wound healing‐promoting effect of ZLY032 combined with single or dual PPARδ/FFA1 knockdown. Down panel: the wound area and wound closure rate of each male mice at varying time points. ** p < 0.01 versus siNC‐m (0.1% DMSO); # p < 0.05, ## p < 0.01, ### p < 0.001 versus siNC‐m+ZLY032 (100 µ m ); n = 4 for each group. Scale bar: 2 mm. (Mean ± SD; two way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) C,D) The blood flow at the wound site was detected by Doppler blood flow detector on day 8 after the administration of ZLY032 combined with single or dual PPARδ/FFA1 knockdown. * p < 0.05, *** p < 0.001 versus siNC‐m (0.1% DMSO), n = 4. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) E,F) ELISA kits to detect the expression level of IL‐6 and IL‐1β in the wound tissue on the 3rd day of ZLY032 (100 µ m ) administration combined with single or dual PPARδ/FFA1 knockdown. *** p < 0.001 versus siNC‐m (0.1% DMSO); ## p < 0.01, ### p < 0.001 versus siNC‐m (0.1% DMSO)+ZLY032 (100 µ m ); n = 6. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032 exhibits antimicrobial activities against S. aureus . A–C) The inhibiting effects of ZLY032 on S. aureus (ATCC 29213, ATCC 6538) and MRSA (ATCC 43300) at the concentrations of 25, 50, and 100 µ m . * p < 0.05 versus CTRL (0.1% DMSO), *** p < 0.001 versus CTRL, n = 4. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) D) To explicit the minimum inhibitory concentration (MIC) of ZLY032 on S. aureus (ATCC 6538). *** p < 0.001 versus CTRL, n = 6. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) E) Time dependent inhibiting of S. aureus (ATCC 6538) by ZLY032 at 50, 100, and 200 µ m . * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL (0.1% DMSO), ## p < 0.01, ### p < 0.001 versus CTRL (0.2% DMSO), n = 5. (Mean ± SD; two‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) F) In vitro clarification assay to verify the inhibitory effect of ZLY032 on S. aureus (ATCC 6538) at the concentrations of 50, 100, and 200 µ m . G) Using scanning electron microscope to demonstrate the damage effect of ZLY032 on S. aureus (ATCC 6538). H,I) The effect of ZLY032 on the activity of S. aureus (ATCC 6538) was determined by crystal violet staining. ***p < 0.001 versus CTRL (0.1% DMSO); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) J) Experiment flowchart of ZLY032 against S. aureus (ATCC 6538). K,L) The effects of ZLY032 on the colony formation of S. aureus (ATCC 6538) after treating by ZLY032 (50 and 100 µ m ) or Penicillin G (50 and 100 µ m ) for 12 and 24 h. ***p < 0.001 versus CTRL (0.1% DMSO)‐12 h, ### p < 0.001 versus CTRL (0.1% DMSO)‐24 h; n = 5 for each group. (Mean ± SD; two‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) M,N) Determination of the MBC of ZLY032 against S. aureus (ATCC 6538) by colony formation assay. n = 3. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Concentration Assay, In Vitro, Clarification Assay, Microscopy, Activity Assay, Staining, Colony Assay

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032 prompted MRSA‐infected wound healing. A) The inhibiting effects of ZLY032 on MRSA (USA300). ***p < 0.001 versus CTRL (0.1% DMSO); n = 4 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) B) The effects of Penicillin G on MRSA (USA300). *p < 0.05, ***p < 0.001 versus CTRL (0.1% DMSO), n = 6. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) C,E) The effect of ZLY032 on colony formation of MRSA (USA300) after treatment with ZLY032 (50 and 100 µ m ) or Penicillin G (50 and 100 µ m ) for 12 h. ** p < 0.01, *** p < 0.001 versus CTRL (0.1% DMSO). # p < 0.05 versus Penicillin G (50 µ m ), & p < 0.05 versus Penicillin G (100 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) D,F) The effect of ZLY032 and Penicillin G on the activity of MRSA (USA300) was determined by crystal violet staining. * p < 0.05, *** p < 0.001, versus CTRL (0.1% DMSO), &&& p < 0.001 versus Penicillin G (100 µ m ); n = 7 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) G,H) Determine the MBC of ZLY032 against MRSA (USA300) by colony formation assay. n = 3. I) Flowchart of MRSA infected wound model in male mice. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) J–L) The wound healing‐promoting effect of ZLY032 MRSA infected wound model in mice. Penicillin G was acted as positive control. * p < 0.05, *** p < 0.001 versus infection; # p < 0.05 versus Penicillin G; n = 5 for each group. Scale bar: 2 mm. (Mean ± SD; two‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) M) Representative images of bacterial colony in wound fluid and the amount of MRSA in the wound tissue on day 3. *** p < 0.001 versus CTRL (0.1% DMSO); # p < 0.05 versus Penicillin G (100 µ m ); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Infection, Activity Assay, Staining, Colony Assay, Positive Control

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: Target identification of ZLY032 in S. aureus . A) Flowchart of RNA sequencing in ZLY032‐treated S. aureus . B) Gene expression changes in S. aureus given ZLY032 treatment. C) GO analysis of differentially expressed genes. D) KEGG analysis of differential genes. E) Differential gene expression analysis screened the genes for significant changes in arginine metabolism and synthesis as well as polysaccharide biosynthesis process after ZLY032 treatment. F) qRT‐PCR detection for the effect of ZLY032 (100 µ m ) on the expression of the screened genes including argH, argJ, argC, argB, argF, arcA, arcC, arcD, sdaA, and ureC. ***p < 0.001 versus CTRL (0.1% DMSO); n = 3–6 for each group. (Student t ‐test for comparisons between two groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Drug discovery, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ASAL is the direct target of ZLY032 in S. aureus . A) Diagram of arginine synthesis in S. aureus . B) ELISA kit to detect the effect of ZLY032 on the expression level of ASAL at 50 and 100 µ m . ***p < 0.001 versus CTRL (0.1% DMSO); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) C) Arginine kit to detect the effect of ZLY032 on the level of arginine at 50 and 100 µ m . **p < 0.01 versus CTRL (0.1% DMSO); n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) D) The effect of ZLY032 on MRSA (USA300) after administration of l ‐arg ( l ‐arginine) at concentrations of 0.1, 0.5, 1, and 2.5 m m . **p < 0.001, ***p < 0.01 versus CTRL. ## p < 0.01, ### p < 0.001 versus ZLY032; n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) E) The construction process of stabilized argH overexpression strains. F) ELISA kit to detect the expression level of ASAL in argH overexpressing strains. ***p < 0.001 versus NC. n = 6 for each group. (Student t ‐test for comparisons between two groups.) G) The inhibiting effect of ZLY032 on the strain of MRSA (USA300) with argH overexpression at 50 and 100 µ m . *p < 0.05, **p < 0.01, ***p < 0.001 versus CTRL (0.1% DMSO). n = 6 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) H) Cellular thermal shift assays (CESTA) to detect the direct binding of ZLY032 on ASAL. ***p < 0.001 versus CTRL (0.1% DMSO); n = 3 for each group. (Mean ± SD; ordinary one‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups.) I) Molecular docking of ZLY032 with ASAL. Hydrogen bonds are shown by blue dashed lines, halogen bonds are shown by orange dashed lines, and π–cation interactions are shown by yellow dashed lines. J) Molecular dynamics simulations at the active pocket with 6IG5. RMSD variation curves of the complexes during 20 ns kinetic simulations. K) RMSF values of amino acid residues in the complex system. L) Number of hydrogen bonds between ZLY032 and 6I5G. M) Structures of the complexes of ZLY032 with 6IG5 at 5, 10, 15, and 20 ns. N) Free energy landscape (FEL) plotted between RMSD and gyration coordinates and the energy minimum conformational state of ZLY032. O) SPR assay to determine the direct interaction between ZLY032 and ASAL (from S. aureus ).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Binding Assay, SPR Assay

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: ZLY032‐loaded microneedles promote wound healing in rabbits. A) Microneedle preparation and animal experimental procedures. B) Observation of microneedle morphology. C) Scanning electron microscope observation of microneedle. D) Methylene blue staining to characterize the skin penetration effect of microneedles. E) Microneedle dissolution experiment at general skin. F) Microneedle dissolution experiment at wound area. G) Left panel: representative photographs of time‐dependent wound healing and ZLY032‐loaded microneedles promoting wound healing in rabbits. Empty‐loaded microneedles were used as positive controls. Middle and right panels: wound area and wound closure rate of each rabbit at different time points. **p < 0.01, ***p < 0.001 versus CTRL (0.1% DMSO). # p < 0.05, ## p < 0.01 versus NC microneedle; n = 4 for each group. (Mean ± SD; two‐way ANOVA followed by Tukey's multiple comparisons test among multiple groups).

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques: Microscopy, Staining, Dissolution

Journal: Advanced Science

Article Title: The Multitarget Compound ZLY032 Achieves Treatment of Chronic Wounds

doi: 10.1002/advs.202503098

Figure Lengend Snippet: Multifunctional role of ZLY032 in wound healing promotion. At treating level, topical application of ZLY032 effectively accelerated the wound healing processes in both mice and rabbits with low toxicity by promoting angiogenesis, inhibiting inflammation and inhibiting S. aureus . At molecular level, ZLY032 acts by targeting PPARδ, FFA1, and ASAL in both eukaryotic and prokaryotic cells. At application level, the microneedles containing ZLY032 can reduce the number of doses and promote wound healing in rabbits.

Article Snippet: Remarkably, ZLY032 showed concentration‐dependent antibacterial activity against S. aureus (ATCC 29213, ATCC 6538), and MRSA (ATCC 43300) compared with the control group and the inhibition rate is higher than 90% at the concentration of 100 μ m ( Figure 4 A–C ; Figure , Supporting Information).

Techniques:

Journal: Bio-protocol

Article Title: Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins

doi: 10.21769/BioProtoc.2769

Figure Lengend Snippet: Samples are kept anaerobic using a double vial system in which all reaction components are added to a small screw cap vial, which are sealed anaerobically in a slightly larger clear glass crimp vial. Protein is injected into the reaction mixture using gas tight Hamilton syringes to initiate the reaction. At designated time points, 10 μl of sample is extracted from the reaction mixture and added to an aerobic quench solution, containing formic acid and porcine pepsin. The acid not only locks in any deuterium that has exchanged onto the protein, but also serves to partially denature the protein complex, making it more accessible to pepsin digestion. Additional unfolding occurs upon exposure to oxygen. After 2 min of digestion, samples are flash frozen in liquid N2 and stored at −80 °C until data acquisition on Agilent 6538 Q-TOF. After acquisition, data is processed to display deuterium uptake over time in the form of uptake curves. Deuterium incorporation can be localized onto the structure of a protein by mapping the deuterium incorporation onto a 3D structure of the protein (if one is available).

Article Snippet: After 2 min of digestion, samples are flash frozen in liquid N 2 and stored at −80 °C until data acquisition on Agilent 6538 Q-TOF.

Techniques: Injection