Discovery And Validation Of The Specific Target KRT1 Of LM49 Against The Hypoxia/reoxygenation Injury On Cardiomyocytes

Objective:Benzophenone-based halophenol compound 5,2’-dibromo-2,4’,5’-trihydroxy-diphenyl-methanone（LM49）had significant protective effects on Na₂S₂O₄-induced hypoxia/reoxygenation injury in H9c2 cells and myocardial ischemia-reperfusion injury in rats,but the targets of LM49 are still not clear.In this paper,we designed and synthesized probes based on the structure of LM49.The target proteins of LM49 were labeled,enriched and analyzed by activity-based protein profiling（ABPP）.For the potential target keratin 1（KRT1）,the binding,signaling pathway and activity verification were carried out to prove that LM49 directly interact with KRT1 and affect the downstream Notch1 signaling pathway,and exert protective effects on cardiomyocytes.Methods:1.The LM49 molecule probes were designed and synthesized,and the structures were confirmed by ESI-MS,¹H-NMR,¹³C-NMR,¹H,¹H-NOESY,HMBC.2.The hypoxia-reoxygenation（H/R）model of H9c2 cells was established by Na₂S₂O₄oxygen consumption method.Cell viability and lactate dehydrogenase（LDH）content were detected to compare the protective effects of probes against H/R injury in H9c2 cells.Moreover,LPS induced inflammation in RAW264.7 macrophages.The concentration of NO and TNF-αwere detected to compare the anti-inflammatory activity of probes on RAW264.7 macrophages.3.Molecule probes were first incubated with living cells and linked to the fluorophore rhodamine by click-chemistry.The binding proteins were then visualized with SDS-PAGE.Screened probe for enrichment experiment and optimized experimental methods.LM49-P2 was incubated with RAW264.7 macrophages and linked to the biotin by click-chemistry,then the probe–protein complexes were enriched through affinity purification.The protein targets were then identified with LC-MS/MS.The results showed that KRT1 is the target protein with high confidence.4.Molecular docking was carried out to clarify the binding mode between LM49,LM49-P2 and KRT1 at the molecular level.The interaction between LM49and KRT1 was verified by CETSA assay and Pulldown assay.Transfected KRT1si RNA to knockdown KRT1 in H9c2 cells.The effect of KRT1 on LM49-mediated protective effect against Na₂S₂O₄-induced hypoxia/reoxygenation injury was detected by MTT assay and LDH detection.RT-q PCR was employed to further examine the impact of KRT1 on LM49-mediated regulation of Notch1 signal pathway and inflammation-related factors in H9c2 cells.Results:1.Designed and synthesized several LM49 small molecule probes,among them,LM49-P2 and LM49-Z2 significantly inhibited the release of NO and TNF-αin the supernatant of RAW264.7 macrophages,and increased the relative viability of hypoxia-reoxygenated H9c2 cells.LM49-P2 significantly inhibited LDH release in H9c2 cells with hypoxia-reoxygenation-induced injury.2.LM49-P1,LM49-P2 and LM49-Z2 were labeled to specific protein bands,after pre-incubation with LM49,LM49-P2 was not labeled with the specific protein band.Combined with the results of activity experiments,LM49-P2 was selected for subsequent experiments.3.ABPP analysis was performed on RAW264.7 cells.Set up solvent group,LM49-P2 group,LM49 competition group.Through LC-MS/MS analysis,enrichment analysis,and venny analysis,the high-confidence target protein was obtained as KRT1.4.Molecular docking simulation suggested that LM49 and LM49-P2 could bind to the same site of KRT1 by forming stable hydrogen bonds.The result from CETSA assay showed that LM49 could protect KRT1 from temperature dependent degradation inhibit KRT1 degradation induced by heating.The result from Pulldown assay showed that KRT1 could be enriched by LM49-P2,LM49 and LM49-P2competed for binding to KRT1.The results proved that KRT1 is a specific binding protein of LM49.KRT1 silencing had cytoprotective effect,and the cytoprotective effect of LM49 were significantly reduced by RNAi-induced knockdown of KRT1.The results indicated that LM49 exert the cytoprotective effect by inhibiting this target.Furthermore,KRT1 silencing also reduced LM49-induced the regulation of Notch1 signal pathway and inflammation-related factors,indicated that LM49regulates Notch1 signaling pathway and exerts cytoprotective effect by interacting with KRT1.Conclusion:1.LM49 could specifically target to KRT1 protein.2.LM49 could regulate Notch1 signaling pathway and exert protective effect on cardiomyocytes by inhibiting KRT1.