Exploring ZPF And Novel Analogues Targeting COX-2/PPAR-? To Alleviate LPS-Induced ALI/ARDS Based On The STSBPT Strategy

Due to the complexity of multifactorial diseases,single-target drugs do not always show satisfactory therapeutic effects.Multi-target pharmacology is considered to be an important strategy for discovering novel drugs and treating complex diseases.Compared with single target drugs,multi-target drugs simultaneously regulate multiple targets to improve the safety and effectiveness of treatment.Acute lung injury and its severe form acute respiratory distress syndrome(ALI/ARDS)are complex multisystem,multitarget,and high mortality diseases characterized by uncontrolled inflammatory responses,increased alveolar capillary permeability,and pulmonary edema,and there is currently no effective drug targeted treatment for ALI/ARDS.ALI/ARDS is mainly a variety of respiratory diseases affecting livestock and poultry,accompanied by the outbreak of inflammation.Therefore,controlling the occurrence of inflammatory storm from the source has become an important strategy for the treatment and control of ALI/ARDS.Zaltoprofen(ZPF),a selective COX-2 inhibitor,has potent anti-inflammatory effects and is metabolized into a variety of metabolites in humans,which can effectively inhibit COX-2 and cause less damage to the gastrointestinal tract than other NSAIDs,such as indomethacin,praprofen.However,whether ZPF exerts anti-inflammatory effects through its several metabolites and its in-depth mechanism still need further investigation,whether ZPF and its active metabolites have the potential of multi-target drugs,and whether ZPF and its active metabolites can inhibit inflammatory responses by multi-target effects to alleviate ALI/ARDS remain uninvestigated.Therefore,in this study,a strategy named “similar topological structure binding properties of protein targets,STSBPT” was developed for the prediction of protein targets of ZPF and its main active metabolites and for the screening of novel analogues based on the parent structure of ZPF and its active metabolites,and multiple protein targets.LPS-induced RAW264.7 cells were used as an inflammatory cell model to investigate the roles and regulatory mechanisms of ZPF and its active metabolites in counteracting LPS induced inflammation in RAW264.7 cells at the cellular level;Using the ALI/ARDS model of LPS induced male C57BL/6 mice,to explore the potential of ZPF,its active metabolites and novel analogues in the treatment of LPS induced ALI/ARDS inflammation at the animal level.The following results were obtained:1.ZPF and active metabolite M2 present high COX-2 inhibitory activity and low COX-1 inhibitory activityThree known metabolites of ZPF,M2,M3 and M5,were successfully synthesized by chemical synthesis method.The COX-1 and COX-2 inhibitor screening kits were used to analyze the COX-2 inhibitory activity of ZPF and its metabolites.The results showed that M2 had stronger COX-2 inhibitory activity and relatively weaker COX-1 inhibitory activity than ZPF,while M3 and M5 had basically no COX-1 inhibitory activity.In addition,application of SYBYL-X2.0,Py MOL as well as the key amino acid site mutation assay to explore the key amino acids involved in the binding of ZPF and metabolites to COX enzyme.From the docking score of molecular docking,the potential of ZPF and metabolites to bind COX enzyme was found to be consistent with the results of the COX inhibitor screening.From the docking results and the site mutation assay verification,Arg120,Tyr355,and Ser530 were found to be the key amino acids residues involved in the binding of ZPF and M2 to COX-2.2.Prediction of PPAR-? as another key anti-inflammatory target of ZPF and M2 based on the STSBPT strategyThis study proposes a strategy for STSBPT from the multiple perspectives of the spatial density of the active pocket involved in receptor ligand binding,the spatial directionality of the key AAR involved in receptor ligand binding,and the pharmacological activities of parent drugs and metabolites.Applied the STSBPT strategy to screen antiinflammatory proteins related to the COX-2 target pathway,and successfully predicted that PPAR-? might be another key anti-inflammatory target of ZPF and M2.Further validation using the CETSA assay and DARTS assay found that ZPF and M2 have the potential to bind to COX-2 and PPAR-?,indicating that ZPF and M2 have the function of dual targets of COX-2 and PPAR-?.3.ZPF and M2 alleviated LPS induced inflammatory response in RAW264.7 cells via MAPKs-PPAR-?/NF-?B signalling pathwayUsing LPS induced RAW264.7 cells as an inflammatory cell model,present study found that ZPF and M2 could inhibit the expression of inflammation related genes and proteins(such as i-NOS,COX-2,PPAR-?,IL-1?,TNF-?)in LPS induced RAW264.7 cells to various degrees.Using nucleus and cytoplasm fractionation assay and immunofluorescence assay,present study found that ZPF and M2 could inhibit the nuclear translocation of NF-?B p65 and activate the nuclear translocation of PPAR-?,thereby inhibiting LPS-induced inflammatory response in RAW264.7 cells.GW9662,an inhibitor of PPAR-?,was applied to explore the association between PPAR-? and NF-?B p65,and this study found that the presence of GW9662 could abolish the inflammatory response of ZPF and M2 against LPS induced RAW264.7 cells to a certain extent,mainly reflected in the ability of GW9662 to counteract the nuclear translocation of PPAR-? as well as promote the nuclear translocation of NF-?B p65.Using LPS-induced RAW264.7 cells as an inflammatory cell model found that ZPF and M2 can inhibit the phosphorylation status of the three subtypes of MAPKs(ERK1/2,p38 MAPK and JNK)to varying degrees.Using the signalling pathway inhibitors of MAPKs,p38 inhibitor(SB203580)and ERK1/2inhibitor(PD98059),present study found that ZPF and M2 inhibited the occurrence of LPS induced inflammatory response differentially,which is reflected in M2 focused on the regulation of p-p38 pathway and the activation of PPAR-? to exert anti-inflammatory effect,while ZPF focused on the regulation of p-ERK pathway and then anti-inflammatory effect.In conclusion,both ZPF and M2 can play a role in the expression of COX-2 and i-NOS through MAPKs-PPAR-?/NF-?B signalling pathway,and thus play a role in counteracting LPS-induced inflammatory response in RAW264.7 cells.4.Screening of novel analogues with the potential of dual anti-inflammatory targets of COX-2 and PPAR-? based on the STSBPT strategyBased on the parent structure of ZPF and M2 and the principle of chemical hybridization,STSBPT strategy was used to screen novel analogues with the dual antiinflammatory target potential of COX-2 and PPAR-?,and successfully screened novel analogues D63,E63,JMC2,JMC5 and JMC6 with dual anti-inflammatory target potential of COX-2 and PPAR-?.CCK-8 assay was used for evaluating the cytotoxicity of RAW264.7 and the cells induced by LPS were used as an inflammatory cell model.The results showed that novel analogues have the potential to inhibit the expression of COX-2protein and promote the expression of PPAR-?,and the effect of novel analogues on COX-2 and PPAR-? was stronger than that of ZPF and M2.5.ZPF,M2 and novel analogues can alleviate the inflammatory damage of ALI/ARDS induced by LPS in male C57BL/6 miceThe inflammatory injury of ALI/ARDS induced by LPS in male C57BL/6 mice was used as an animal model to explore the therapeutic effects of ZPF,M2 and novel analogues on the inflammatory injury of ALI/ARDS induced by LPS.The results showed that LPS could significantly induce lung edema in mice,while ZPF,M2 and novel analogues could reduce the occurrence of LPS-induced pulmonary edema in different degrees.In this study,histopathological observation and immunohistochemical analysis found that ZPF,M2 and novel analogues can reduce the inflammatory damage in lung caused by LPS to varying degrees,and ZPF,M2 and novel analogues could alleviate inflammatory injury effects of LPS-induced ALI/ARDS in male C57BL/6 mice via COX-2 and PPAR-?-dependent manners.From the perspective of the feasibility of STSBPT strategy and the dual targets of COX-2 and PPAR-?,this study commits to explore the multiple anti-inflammatory targets,anti-inflammatory mechanisms and potential of ZPF,active metabolite M2 and novel analogs in the treatment of ALI/ARDS,so as to realize the strategy of "one drug,multiple targets,one disease" and lays a theoretical foundation to the prevent and treat the multisystemic inflammatory diseases,especially ALI/ARDS,in animal husbandry industry.