Discovery Of Novel Natural Inhibitors Of Caspase-1 With Anti-pyroptosis Activity In Vitro And In Vivo

BackgroundPyroptosis,also known as cell inflammatory necrosis,is a programmed cell death.Compared to apoptosis,pyroptosis occurred faster,with the release of a large number of inflammatory cytokines.Pyroptosis is widely involved in the occurrence and development of diseases.The development of pyroptosis inhibitors has attracted more and more researchers’ attention.The activation pathways of pyroptosis include Caspase-1-dependent canonical pathway and Caspase-4/-5/-11 dependent noncanonical pathway.In the canonical pathway,the activated Caspase-1 leads to the cleavage of GSDMD,forming GSDMD-N,and inducing membrane perforation.At the same time,activated Caspase-1 cleaves pro-IL-1β and pro-IL-18 to form active IL-1βand IL-18,which are released extracellular to recruit inflammatory cells,thereby amplifying the inflammatory response.Caspase-1 inhibitors,such as VX-765,have anti-pyroptosis activity in vitro and in vivo,alleviating a variety of pyroptosis diseases.Therefore,Caspase-1 is a target for screening pyroptosis inhibitors.Natural small molecule compounds have the advantages of low immune response and good pharmacokinetics in drug development,and have irreplaceable value.Many drugs that have been approved and tested in clinical trials are derived from natural products,such as aspirin,artemisinin and curcumin.At present,Caspase-1 inhibitors are mainly synthetic peptides and peptide-based compounds,while Caspase-1 inhibitors obtained from natural products are rarely reported.Therefore,small molecule inhibitors of Caspase-1 based on natural products with anti-pyroptosis activity in vitro and in vivo need to be studied and reported.ObjectiveWith Caspase-1 as the target,inhibitors were screened in natural products,and anti-pyroptosis activity was studied in cell model and animal model,so as to discovery natural products with anti-pyroptosis activity in vitro and in vivo.Methods1.Protein expression and purificationCaspase-1 and other recombinant proteins were expressed by Escherichia coli protein expression system,and the recombinant proteins were purified by Ni-NTA Chromatography,ion exchange chromatography and gel filtration chromatography.2.Screening of Caspase-1 inhibitors based on 2526 natural productsThe high-throughput screening system of "Caspase-1+Ac-YVAD-pNA+compounds" was established based on recombinant Caspase-1 protein and substrate Ac-YVAD-pNA,and the "Caspase-1+GSDMD+ compound" verification system was established based on recombinant Caspase-1 and GSDMD protein.3.Experiments on protein-small molecule interactionsThe affinity of the compounds with Caspase-1 protein was analyzed by Surface plasmon resonance(SPR),Differential Scanning Fluorimetry(DSF)and Cellular thermal shift assay(CETSA).The molecular weight of Caspase-1 protein was determined by HPLC-Q-TOF-MS.4.Evaluation of cell activity of demethylzeylasteral and ginkgolic acid C15:1The pyroptosis model of bone marrow derived macrophages(BMDM)were established by LPS and ATP.The activation of Caspase-1 and GSDMD was detected by WB,the release of inflammatory factors was detected by ELISA,the cell death rate was detected by Propidium iodide(PI)staining,and ASC speck was detected by cellular immunofluorescence.In BMDM cells,Caspase-1 was silenced by siRNA.The pyroptosis model was established in cells transfected with Negative siRNA and Caspase-1 siRNA,respectively,and the activities of demethylzelaminaldehyde and ginkgolic acid C15:1 were investigated.5.Evaluation of the in vivo activities of demethylzeylasteral and ginkgolic acid C15:1Mice model of acute lung injury was established by LPS intratracheal instillation.In the animal experiment of demethylzeylasteral,C57BL/6J mice were randomly divided into 12 mice per group,and the groups were as follows:Control,ALI model,ALI+demethylzeylasteral(3.75 mg/kg),ALI+demethylzeylasteral(7.5 mg/kg),ALI+ demethylzeylasteral(15 mg/kg),ALI+VX-765(50 mg/kg)and ALI+dexamethasone(5 mg/kg).Demethylzeylasteral was prepared with 0.5%CMCNa and administrated intragastrically.VX-765 was prepared with 20%Cremophor solution and administered intraperitoneally.Dexamethasone injection was diluted with normal saline and administered intraperitoneally.Before modeling,the drug was given once a day for 7 days.In the animal experiment of ginkgolic acid,C57BL/6J mice were randomly divided into 12 mice per group,and the groups were as follows:Control,ALI model,ALI+ginkgolic acid(10 mg/kg),ALI+ginkgolic acid(20 mg/kg),ALI+ginkgolic acid(30 mg/kg),ALI+ VX-765(50 mg/kg),ALI+dexamethasone(5 mg/kg).The ginkgolic acid was dissolved by DMSO(10%),then PEG300(40%),Tween-80(5%)and normal saline(45%)were added successively,and administered intraperitoneally.HE staining of lung tissue and the levels of total protein,albumin and LDH in bronchoalveolar lavage fluid were used to evaluate the ameliorative effects of demethylzeylasteral and ginkgolic acid on lung injury.The activation of Caspase-1,the cleavage of GSDMD and the release of inflammatory factors were detected by WB,ELISA and immunohistochemistry,to verify the anti-pyroptosis activities of demethylzeylasteral and ginkgolic acid in vivo.Results1.Protein expression and purificationInflammatory Caspases(Caspase-1,Caspase-4 and Caspase-5),non-inflammatory Caspases(Caspase-3 and Caspase-6)and GSDMD were obtained with high purity by protein expression and purification.The results of protein activity showed that the purified recombinant proteins had good activity,which could be used for compounds screening and activity verification experiments.2.Screening of Caspase-1 inhibitors based on 2526 natural productsBased on the high throughput screening system of "Caspase-1+Ac-YVAD-pNA+compounds",2526 natural products were screened and retested,and 24 active compounds were obtained.The activity of 24 compounds was determined by the"Caspase-1+GSDMD+ compounds" verification system,and 7 target compounds were obtained.Next,the affinity between the target compounds and the protein was detected by molecular interaction technology,and the results were as follows:①DSPR results showed that the KD value of denosyl cobalamine and gossypol were greater than 200 μM and 50 μM,respectively,showing weak affinity.②SPR results showed that the KD value between ginkgolic acid C15:1 and Caspasae-1 was 4.83 μM,and the KD value of positive control VX-765 was 11.3 μM,indicating that ginkgolic acid C15:1 had a higher affinity for Caspase-1 than VX-765.③DSF and CETSA were used to investigate the effect of demethylzeylasteral on protein thermal stability.The DSF results showed that the Tm value of Caspase-1 increased from 44 ℃ to 53.3 ℃ after incubation with demethylzeylasteral.At the same time,CETSA results based on cell lysates also showed that demethylzeylasteral increased the thermostability of Caspase1.In the SPR experiment,demethylzeylasteral failed to eluted on the chip,suggesting that it may covalently bind to Caspase-1.Therefore,the molecular weight of the protein was determined by HPLC-Q-TOF-MS,and the results showed that the molecular weight of the protein increased after co-incubation with demethylzeylasteral,which proved that demethylzeylasteral was a covalent inhibitor of Caspase-1.The binding kinetic parameters showed that the Kinact/Ki of demethylazelamal on Caspase-1 was 1370.86 ± 74.08 M-1min-1.The results of ginkgolic acid analogues showed that the IC50 value of anacardic acid was 4.959 ± 0.419 μM,which was similar to that of ginkgolic acid,while the IC50 of cardanol and salicylic acid were both greater than 200 μM.The Coomassie bright blue gel was consistent with the results of enzyme activity.This shows that the activity of ginkgolic acid requires the coexistence of carboxyl group and carbon chain in benzene ring.The results of demethylzeylasteral analogues showed that the IC50 value of celastrol was 18.02 ±1.48 μM,and the IC50 value of pristimerin was 168.1 ± 23.6 μM,both of which were inferior to that of demethylzeylasteral(IC50=7.255 ± 0.371 μM).At the same time,the coomassie blue gel of GSDMD showed the same results,indicating that carboxyl group is one of the key functional groups that inhibit Caspase1 activity of demethylzeylasteral and celastrol.The aldehyde group on the benzene ring has strong electrophilic ability and can react with the sulfhydryl group on cysteine,which is the active group covalently bound to Caspase-1.In addition,the IC50 of Caspase-1,Caspase-4,Caspase-5,Caspase-3 and Caspase6 showed that ginkgolic acid and demethylzeylasteral were pan-Caspase inhibitor.3.Evaluation of cell activity of demethylzeylasteral and ginkgolic acidThe WB results of the cell supernatant showed that there was almost no Caspase1 p20 in the cell supernatant of the normal cells.Under the stimulation of LPS and ATP,a large amount of Caspase-1 was activated in the cells of the model group to form Caspase-1 p20,which was released into the medium.However,the level of Caspase-1 p20 in the supernatant was significantly decreased after the treatment of demethylzeylasteral and ginkgolic acid respectively.Meanwhile,FAM-FLICA Caspase-1 showed that both demethylzeylasteral and ginkgolic acid could reduce the fluorescence level.Therefore,the activation of Caspase-1 was inhibited by demethylzeylasteral and ginkgolic acid.The WB results of cell lysate showed that there was almost no GSDMD-N in the normal group,but GSDMD-N was activated significantly in the model group under the stimulation of LPS and ATP,and a large amount of GSDMD-N was produced.The level of GSDMD-N decreased after treatment with demethylzeylasteral and ginkgolic acid.At the same time,demethylzeylasteral and ginkgolic acid also reduced IL-1β,IL-18 and LDH.Moreover,PI staining showed that demethylzeylasteral and ginkgolic acid inhibited cell mortality.In addition,the results of immunofluorescence showed that both demethylzeylasteral and ginkgolic acid had no significant effect on the formation of ASC speck.In BMDM cells,the expression of Caspase-1 was significantly reduced after transfection with Caspase-1 siRNA compared to cells transfected with Negative siRNA.In the cells transfected with Negative siRNA,both 2.5 μM demethylzeylasteral and 5μM ginkgolic acid significantly inhibited the production of GSDMD-N and the release of IL-1β,IL-18 and LDH.However,2.5 μM demethylzeylasteral did not inhibit the activation of GSDMD in Caspase-1 siRNA cells,nor did it inhibit the release of IL-1β,IL-18 and LDH.Although GSDMD-N,IL-1β and IL-18 decreased with 5 μM ginkgo acid,the difference was not statistically significant.4.Evaluation of the in vivo activities of demethylzeylasteral and ginkgolic acidHE staining results showed that the lung tissue of mice in the model group presented serious pathological morphology of alveolar interstitial edema,alveolar wall thickening and neutrophil infiltration.Compared with the Control group,the total protein,albumin and LDH in the BALF fluid of mice in the ALI model were significantly increased,indicating that the ALI model was successfully constructed in this experiment.Demethylzeylasteral improved lung tissue lesions in mice and reversed abnormal increases in total protein,albumin and LDH induced by lung injury,in a dosedependent manner.The amelioration effect of demethylzeylasteral on lung injury was statistically different from that of VX-765 and dexamethasone.Similarly,ginkgolic acid also alleviated lung injury and total protein,albumin and LDH,showing a dosedependent manner,and the activity of ginkgolic acid C15:1 was statistically different from that of VX-765 and dexamethasone.WB results showed that Caspase-1 p45 and Caspase-1 p20 could not be detected in BALF of Control group,while Caspase-1 p45 and Caspase-1 p20 protein levels increased sharply in ALI model group.Consistent with the results of BALF,WB and immunohistochemical results of lung tissue also showed high expression of Caspase-1 p45 and Caspase-1 p20 in the model group,indicating that the established ALI model had obvious Caspase-1 activation characteristics and was suitable for evaluating the activity of Caspase-1 inhibitor.Compared with model group,Caspase-1 p45 and Caspase-1 p20 in BALF and lung tissue were significantly decreased in demethylzeylasteral and ginkgolic acid groups,and the inhibitory effect of 15 mg/kg demethylzeylasteral and 30 mg/kg ginkgolic acid C15:1 on Caspase-1 was statistically different from that of the positive control VX-765(50 mg/kg).Consistent with the results of Caspase-1,almost no GSDMD-N was detected in BALF in the Control group,while a sharp increase of GSDMD-N was detected in the model group,indicating obvious pyroptosis in ALI model.Compared with the model group,lower levels of GSDMD-N were detected in the demethylzeylasteral,ginkgolic acid and VX-765 groups.IL-1β and IL-18 in BALF were quantitatively determined by ELISA.The results showed that IL-1β and IL-18 were significantly increased in the model group under LPS induction(P<0.001).However,the abnormal increase of IL-1β and IL-18 was reversed in the administration groups of demethylzeylasteral,ginkgolic acid,VX-765 and dexamethasone,and the activity of 15 mg/kg demezeralaldehyde and 30 mg/kg ginkgolic acid C15:1 was statistically different from those of VX-765 and dexamethasone.Conclusions1.Ginkgolic acid C15:1 and demethylzeylasteral are Caspase-1 inhibitors,and demethylzeylasteral is a covalent inhibitor.2.In BMDM pyroptosis model,ginkgolic acid C15:1 and demethylzeylasteral exert anti-pyroptosis effects by inhibiting Caspase-1 activation.3.In the LPS-induced ALI model,both ginkgolic acid C15:1 and demethylzeylasteral significantly improved lung tissue lesions and inhibited pyroptosis.