Discovery of Balasubramide Derivative with Tissue-Specific Anti-Inflammatory Activity Against Acute Lung Injury by Targeting VDAC1

Abstract

Macrophage-mediated inflammatory responses including pyroptosis are involved in the pathogenesis of sepsis and acute lung injury (ALI), for which there are currently no effective therapeutic treatments. The natural product (+)-Balasubramide is an eight-membered lactam compound extracted from the leaves of the Sri Lanka plant Clausena Indica and has shown anti-inflammatory activities, but its poor pharmacokinetic properties limit its further application for ALI. In this study, a compound (+)3C-20 is discovered with improved both pharmacokinetic properties and anti-inflammatory activity from a series of (+)-Balasubramide derivatives. The compound (+)3C-20 exhibits a markedly enhanced inhibitory effect against LPS-induced expressions of pro-inflammatory factors in mouse macrophages and human PBMCs from ALI patients and shows a preferable lung tissue distribution in mice. (+)3C-20 remarkably attenuates LPS-induced ALI through lung tissue-specific anti-inflammatory actions. Mechanistically, a chemical proteomics study shows that (+)3C-20 directly binds to mitochondrial VDAC1 and inhibits VDAC1 oligomerization to block mtDNA release, further preventing NLRP3 inflammasome activation. These findings identify (+)3C-20 as a novel VDAC1 inhibitor with promising therapeutic potential for ALI associated with inflammation.

Keywords: NLRP3 inflammasome; VDAC1; acute lung injury; balasubramide derivative; macrophage.

Figure 1

Discovery of balasubramide derivative (+)3C‐20 with strong anti‐inflammation in macrophage. A) Sources of natural product derivatives (+)3C‐20. B, C) ELISA analysis of TNF‐α secretion in supernatants of LPS (100 ng mL⁻¹)‐activated RAW264.7 and BMDMs treated with various concentrations of (+)3C‐20. n = 3. D) Time‐dependent manners of (+)3C‐20 in inhibiting LPS‐induced TNF‐α release in BMDMs. Cells were incubated with (+)3C‐20 (5 µmol L⁻¹) followed by treatment of 100 ng mL⁻¹ LPS for different periods of time and TNF‐α in supernatant was detected by ELISA. n = 3. E) The qRT‐PCR analysis of Tnf‐α, Il‐1β, Il‐6 and Ptgs2 mRNA expression of LPS‐stimulated RAW264.7 treated with or without (+)3C‐20. n = 4. F) The qRT‐PCR analysis of TNFA, IL1B, IL6, and PTGS2 mRNA expression of LPS‐stimulated THP‐1 cells treated with or without (+)3C‐20. n = 4. G–I) After pretreated with compound (10 µM) or vehicle (DMSO) for 3 h, BMDMs were stimulated with LPS (100 ng mL⁻¹) for 3 h followed by ATP (5 mM, 45 min), Nigericin (10 µM, 1 h), or MSU (600 µg mL⁻¹, 6 h) treatment. n = 3. G) Cell morphology, scale bar = 50 µm. H,I) LDH cytotoxicity. Results are expressed as mean ± SD; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus Control group; ^#p< 0.05, ^##p< 0.01, ^### p < 0.001 versus LPS group, LPS plus ATP group, LPS plus Nig group or LPS plus MSU group.

Figure 2

(+)3C‐20 attenuates inflammatory response in acute lung injury induced by sepsis. A) Experimental design to study the effects of (+)3C‐20 against sepsis‐induced ALI. B) Lung index of sepsis‐induced ALI mice administrated with (+)3C‐20 or vehicle. n = 7. C) ELISA analysis of TNF‐α protein in serum from sepsis‐induced ALI mice administrated with (+)3C‐20 or vehicle. n = 8–10. D) The qRT‐PCR analysis of pro‐inflammatory cytokines (Tnf‐α, Il‐1β, Il‐6, and Inos) expression of lung tissue of mice with lung injury administrated with (+)3C‐20 or vehicle. n = 7–8. E,F) H&E staining and histological score of lung tissues from sepsis‐induced ALI mice administrated with (+)3C‐20 or vehicle. Scale bar, 50 µm, n = 3. G) The pharmacokinetic analysis of (+)3C‐20 in mice. H) Organ coefficients of mice after 28 days of administration with (+)3C‐20 or vehicle. n = 7–8. Results are expressed as mean ± SD; ^***p< 0.001 versus the Vehicle group; ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus the LPS group.

Figure 3

(+)3C‐20 significantly ameliorates acute lung injury of mice induced by intratracheal infusion of LPS. A) Experimental design to study the effects of (+)3C‐20 against intratracheal instillation of LPS‐induced ALI in mice. B) Lung index of intratracheal instillation of LPS‐induced ALI mice administrated with (+)3C‐20 or vehicle. n = 10‐12. C) ELISA analysis of TNF‐α protein in lung tissues from ALI mice induced by instillation of LPS and administrated with (+)3C‐20 or vehicle. n = 10‐12. D) The qRT‐PCR analysis of pro‐inflammatory cytokines (Tnf‐α, Il‐1β, Il‐6, Inos, and Ptgs2) expression in lung tissues of mice with lung injury administrated with (+)3C‐20 or vehicle. n = 10–12. E,F) H&E staining and histological score of lung tissues from intratracheal instillation of LPS‐induced ALI mice administrated with (+)3C‐20 or vehicle. Scale bar, 50 µm, n = 9. G) Survival curve of mice treated with (+)3C‐20 or dexamethasone subjected to intratracheal instillation of LPS followed by observation for 72 h. n = 9. H–J) Wet/dry ratio of lung tissues, TNF‐α protein, and the total protein in BALF were measured after mice were treated with (+)3C‐20 (50 mg kg⁻¹) and stimulated with LPS (20 mg kg⁻¹, i.t.). n = 7‐8. K–M) The ratio (%) of cell numbers to total cells per mouse, CD45⁺CD11b⁺F4/80⁺ macrophage, CD45⁺CD11b⁺Ly6G⁺ neutrophils and CD45⁺CD11b⁺Ly6C⁺ monocytes in BALF, n = 6–7. Results are expressed as mean ± SD; ^*p< 0.05, ^***p< 0.001 versus Vehicle group; ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus the LPS group.

Figure 4

Target identification of (+)3C‐20 by activity‐based protein profiling (ABPP). A) Schematic illustration of target protein capture of (+)3C‐20 in macrophages based on ABPP. B) SDS‐PAGE gels of fluorescence scanning imaging and Coomassie staining in situ. C) Silver staining of enriched protein and immunoblotting with anti‐streptavidin‐HRP antibody based on (+)3C‐20‐probe. D) Venn diagrams showing the proteins with high peptide matching scores (score ≥ 10) that were significantly enriched by photoaffinity probe (+)3C‐20‐probe and further analyzed by LC‐MS/MS.

Figure 5

(+)3C‐20 binds and co‐localizes with VDAC1 in macrophages. A,B) Pull‐down and immunoblotting experiments were used in RAW264.7 cells for the target validation of VDAC1 with the photo affinity probes in vitro and in situ. C) The interaction of Biotin‐(+)3C‐20 with VDAC1 was examined in vitro using rhVDAC1 protein, as well as competition with (+)3C‐20. D) The interaction of Biotin‐(+)3C‐20 with recombinant human VDAC2 was examined in vitro. E) CETSA analysis of intracellular binding between (+)3C‐20 (20 µM) and VDAC1 at different temperatures. F) ITDRF analysis of intracellular binding between VDAC1 and (+)3C‐20 at different concentrations at 57 °C. G) SPR analysis of the interactions between (+)3C‐20 and VDAC1, and the K_D value was ≈3.95 µM. H) Immunofluorescence analysis of co‐localization of Biotin‐(+)3C‐20 with VDAC1 or mitochondria in BMDMs. Scale bars, 10 µm. I) The TNF‐α release level in the supernatant of RAW264.7 cells was detected by ELISA. RAW264.7 cells were treated with DMSO or (+)3C‐20 for 3 h following transduction with siRNA against VDAC1 and then stimulated with LPS for 2 h. n = 4. Results are expressed as mean ± SD; ^***p< 0.001 versus siNC plus LPS group.

Figure 6

(+)3C‐20 blocks VDAC1 oligomerization and inhibits cytosolic mtDNA release and NLRP3 inflammasome activation. A) Immunoblotting analysis of the effect of (+)3C‐20 treatment on the activation of NLRP3 inflammasome in BMDMs. BMDMs pretreated (+)3C‐20 were incubated with LPS (100 ng mL⁻¹) for 3 h and then stimulated with ATP (5 mM) for 45 min. B) Relative cytosolic mtDNA analysis of (+)3C‐20 treatment in BMDMs stimulated with LPS. n = 3. C) Immunofluorescence assay of BMDMs that were co‐stained for 8‐OHdG and Hoechst before and after stimulation with LPS plus the indicated inflammasome activators. Scale bars, 10 µm. D) Western blotting images for VDAC1 oligomerization in BMDMs treated with (+)3C‐20 at different concentrations following LPS stimulation. E) View of the binding between (+)3C‐20 and VDAC1 protein by molecular docking. F,G) Western blotting images and quantification for VDAC1 oligomerization in HEK293T cells transfected with VDAC1 (WT, G021A, Y022A, K028A) mutant expression plasmids. n = 4. H) Anti‐inflammatory efficiency of (+)3C‐20 in BMDMs compared to other VDAC1 oligomerization inhibitors (VBIT‐4, VBIT‐12). n = 3. Results are expressed as mean ± SD; ^**p< 0.01, ^***p< 0.001 versus Control group, ^##p< 0.01, ^###p< 0.001 versus LPS group or DMSO group, ^&&&P< 0.001 versus (+)3C‐20 group.

Figure 7

(+)3C‐20 inhibits the activation of NLRP3 inflammasome by targeting VDAC1 and ameliorates ALI in mice induced by intratracheal instillation of LPS in vivo. A) Experimental design to study whether the therapeutic effect of (+)3C‐20 is dependent on VDAC1 in intratracheal instillation of LPS‐induced acute lung injury in vivo. B,C) H&E staining and histological score of lung tissues from LPS‐induced mice reconstituted of VDAC1‐knockdown macrophages in the presence or absence of (+)3C‐20 treatment (50 mg kg⁻¹). Scale bar, 50 µm. D) Lung index of LPS‐induced mice reconstituted of VDAC1‐knockdown macrophages in presence or absence of (+)3C‐20 treatment. E,F) ELISA analysis of TNF‐α protein in the lung tissue and serum from LPS‐induced mice reconstituted of VDAC1‐knockdown macrophages in the presence or absence of (+)3C‐20 treatment. G–J) The qRT‐PCR analysis of pro‐inflammatory cytokines (Tnf‐α, Il‐1β, Il‐6, and Ptgs2) expression in lung tissues of LPS‐induced mice reconstituted of VDAC1‐knockdown macrophages in the presence or absence of (+)3C‐20 treatment. K,L) Immunoblot analysis of NLRP3, cleaved caspase‐1 p20 and IL‐1β p17 protein expression in lung tissues from LPS‐induced mice reconstituted of VDAC1‐knockdown macrophages in the presence or absence of (+)3C‐20 treatment. All data are represented as mean ± SD, control groups, n = 3, other groups, n = 5–6. ^**p< 0.01, ^***p< 0.001 versus shNC group; ^#p< 0.05, ^##p< 0.01, ^###p< 0.001 versus LPS group or shNC plus LPS group.

Figure 8

(+)3C‐20 inhibits cytosolic mtDNA release and inflammation in PBMCs from ALI patients. A) The qRT‐PCR analysis of mtDNA release into the cytoplasm in PBMCs isolated from patients with or without (+)3C‐20 (10 µM) treatment. n = 12. B) ELISA analysis of TNF‐α secretion in supernatants of LPS‐activated PBMCs with or without (+)3C‐20 treatment. n = 12. C–G) The qRT‐PCR analysis of pro‐inflammatory cytokines (TNFA, IL1B, IL‐6, and PTGS2) and VDAC1 gene expression of LPS‐activated PBMCs in the presence or absence of (+)3C‐20 (10 µM) treatment. n = 12. H) The correction analysis was performed between the level of cytosolic mtDNA release and the concentration of TNF‐α in PBMCs from ALI patients. n = 12. I) Schematic diagram of the mechanisms by which natural product derivative (+)3C‐20 binds with VDAC1 and regulates the oligomerization of VDAC1, thereby limiting the activation of NLRP3 inflammasome in macrophages and alleviating pulmonary inflammation in acute lung injury. All data are represented as mean ± SD. ^**p< 0.01, ^***p< 0.001 versus Control group; ^##p< 0.01, ^###p< 0.001 versus LPS group.