Identification Of A Novel Heat Shock Protein 90 Family Inhibitor And Study On Its Role In Autophagy Regulation

BACKGROUND AND OBJECTIVEHeat shock protein 90(HSP90)is a highly conserved molecular chaperone.There are four isoforms in vertebrates,including Hsp90? and Hsp90? in cytoplasm,glucose-regulatory protein 94(Grp94)in endoplasmic reticulum and tumor necrosis factor receptor-associated protein 1(Trap-1)in mitochondria.HSP90 is associated with the occurrence of various diseases.HSP90 inhibitors have been used in the treatment of cancer,neurodegenerative diseases,viral and fungal infections and other diseases.Currently,studies on HSP90 inhibitors are focusing on the inhibition of cytoplasmic isoforms Hsp90?/?.Grp94 is located in endoplasmic reticulum and plays a different role compared with Hsp90?/?.Grp94 participates in endoplasmic reticulum protein folding,protein quality control,regulation of Ca2+homeostasis and cellular immune responses.In addition,Grp94 is associated with the occurrence of diseases such as various tumors and glaucoma,and has become a potential target for clinical treatment.Therefore,it is imperative to develop Grp94 isoform selective inhibitorsAs the main pathogenic factor of atherosclerosis,oxidized low-density lipoprotein(oxLDL)induces autophagy in vascular endothelial cells.Autophagy is highly conserved in eukaryotic cells and is a process of transporting excess or potentially toxic substances to the lysosomes for degradation in cells.Autophagy has both beneficial and detrimental effects on cell survival.Basal level autophagy promotes cell survival while excessive activation of autophagy promotes cell death.Therefore,regulation of autophagy homeostasis can inhibit oxLDL-induced vascular endothelial cell damage.Endoplasmic reticulum stress is involved in the regulation of autophagy and induces the expression of Grp94.OxLDL can also induce autophagy by activating endoplasmic reticulum stress and increase Grp94 expression.However,it is still unclear whether Grp94 participates in the regulation of oxLDL-induced endothelial cell autophagy.In addition,dis-regulation of autophagy is associated with the occurrence of cardiovascular diseases,cancers,and neurodegenerative diseases.Vascular endothelial cell damage caused by oxLDL promotes the development of atherosclerosis and plaque instability.Expression of Grp94 increased in advanced atherosclerotic plaques and it is unclear whether Grp94 is involved in the regulation of atherosclerosis yet.During different stages of tumor development,autophagy plays dual roles.Generally,autophagy inhibits tumorigenesis as well as promotes tumor cell growth and drug resistance.The expression of HSP90 in tumor cells has 2-10 fold change than normal cells,and thus it is speculated to associate with the formation and development of tumors.In addition,HSP90 regulates autophagy through its client proteins,including Akt,Beclinl,ULK1 and lysosomal associated membrane protein 2A.HSP90 inhibitors can simultaneously cause degradation of a variety of tumor-associated proteins and inhibit the growth of lung cancer cells,but the regulatory role of HSP90 in lung cancer cell growth and autophagy needs to be further clarified.Coumarin scaffolds drive the development of HSP90 inhibitors.The structural-activity relationship of the compounds indicates that C-4,C-7 and C-8 of coumarin play a key role in HSP90 inhibition.In the pilot study,we synthesized a novelcoumarin-pyrazoline derivative DPB that inhibited the activity of HSP90.By increasing the structural diversity and water solubility of the compounds,we synthesized novel coumarin-pyrazoline derivatives HCP1-HCP6.Among them,HCP1 can inhibit the activity of HSP90,and selectively inhibit Grp94 at low concentrations.Utilizing HCP1,we profiled the regulatory role of Grp94 in oxLDL-induced autophagy of vascular endothelial cells as well as the regulatory role of HSP90 in autophagy of A549 lung cancer cells.In addition,we confirmed the inhibitory effects of HCP1 on atherosclerosis and lung cancer.Therefore,our study further elucidated the regulation mechanisms of HSP90 family members on autophagy and provided promising targets and leverageable insights in the treatment of atherosclerosis and lung cancer.STUDY CONTENTS1.Study of the inhibitory effects of coumarin pyrazoline derivatives on HSP90 activity.2.Study of the inhibition mechanism of oxLDL-induced autophagy in vascular endothelial cells by low-dose HCP1.3.Study of the effects of HCP1 on atherosclerosis in apoE-/-mice.4.Study of the inhibition mechanism of A549 cell growth and autophagy by high-dose HCP1.METHODS1.Immunofluorescence staining to detect the co-localization of small chemical molecule and protein,protein and protein and the distribution of protein in cells.2.Surface plasmon resonance experiment to detect the binding of small chemical molecule to protein.3.Western blot to detect changes in protein levels and phosphorylation of proteins.4.RNA interference and gene knockout to reduce the expression of specific proteins in cells and plasmid transient transfection to increase the expression of specific proteins in cells.5.Site-directed mutagenesis was used to construct Grp94 site 3 mutant overexpression vectors,including c-Myc-Grp94-wt,c-Myc-Grp94-mt1,-mt2,-mt3,and-mt4,which determined the binding sites of HCP1 and Grp94.6.SRB experiment to determine cell viability.7.Co-immunoprecipitation experiment to detect protein-protein interactions.8.ApoE-/-mice fed with high-fat diet to establish animal model of atherosclerosis and detect the effects of HCP1 on atherosclerosis.9.Hoechst 33258 staining and TUNEL staining were used to detect apoptosis.10.Masson trichrome staining to detect collagen content in plaque;immunofluorescence staining to detect the content of smooth muscle cells and macrophages in plaque;in situ gelatin zymography to detect the activity of MMP2/9 in plaque.11.Lactate dehydrogenase kit to measure the activity of LDH in cell supernatant.12.Chicken chorioallantoic membrane model to detect the effects of HCP1 on tumor growth and apoptosis and the effects of HCP1 on angiogenesisRESULTS1.Novel coumarin pyrazoline derivative HCP1 inhibited the activity of HSP90 family members1.1 Coumarin pyrazoline derivatives HCP1-HCP6 inhibited the binding of HSP90 and its antibody in a concentration-dependent manner.Among them,HCP1 exerted the best inhibitory effect1.2 HCP1 co-localized with Grp94 in cells.Surface plasmon resonance analysis showed that HCP1 bound directly to Grp94 with an equilibrium dissociation constant(KD value)of 0.923 ?M.HCP1 inhibited the protein levels of integrin a2 and the transport of TLR9 from endoplasmic reticulum to lysosome.Transfection with Grp94 site 3 mutant c-Myc-Grp94-mtl plasmid inhibited the effect of HCP1 on integrin ?2 protein levels.1.3 HCP1 at low concentrations(1 ?M,2 ?M,5 ?M)did not affect the protein levels of Hsp90?/? client proteins:Akt,NF?B(p65)and Hsp70.HCP1 at high concentration(10 ?M)decreased Akt and NF?B(p65)protein levels.2.HCP1 inhibited oxLDL-induced HUVEC autophagy by inhibiting Grp942.1 OxLDL increased Grp94 protein levels in HUVECs,and HCP1(0.5 ?M,1 ?M,2?M)could inhibited the decrease in HUVEC viability caused by oxLDL.2.2 HCP1(1 ?M,2 ?M)reduced the number of LC3 dots and protein levels of LC3-?induced by oxLDL in HUVECs.Overexpression of c-Myc-Grp94-wt inhibited the effect of HCP1 on LC3-? protein levels.2.3 Grp94 interacted with AMPK,and HCP1 reduced the phosphorylation level of AMPK(Thr172)in HUVECs.Overexpression of c-Myc-Grp94-wt inhibited the effect of HCP1 on phosphorylation of AMPK(Thr 172)2.4 HCP1 increased the phosphorylation levels of mTORC1 downstream targets p70S6K and 4EBP1.AMPK activator Acadesine(AICAR)inhibited the effect of HCP1 on mTORC1 activity.AICAR also inhibited the effect of HCP1 on LC3-?protein levels2.5 HCP1 inhibited oxLDL-induced cleavage of PARP in HUVECs.3.HCP1 promoted atherosclerotic plaque stabilization in apoE-/-mice3.1 HCP1 did not affect body weight and organ coefficient of mice.3.2 HCP1 reduced LC3 dots in plaque endothelium.3.3 HCP1 inhibited apoptosis of endothelium,macrophages and smooth muscle cells in plaque.3.4 HCP1 increased collagen content and smooth muscle cells,decreased the activity of matrix metalloproteinase MMP-2/9,decreased pro-inflammatory M1 macrophages(CD11C+CD68+),increased anti-inflammatory M2 macrophages(CD206+CD68+)in plaque.4.HCP1 inhibited the growth and autophagy of A549 cells4.1 HCP1-HCP6 decreased cell viability of A549 cells in a concentration-dependent manner.Among them,HCP1 possessed the lowest half-inhibitory concentration IC50(?M)value.4.2 HCP1-HCP6 did not affect the activity of LDH in A549 cell culture supernatant.4.3 HCP1 did not affect the growth of HUVECs under normal conditions.4.4 HCP1 increased LC3-? protein levels in a concentration-and time-dependent manner in A549 cells.HCP1 also increased the protein level of autophagy selective substrate p62.The lysosome inhibitor bafilomycin A1 inhibited the effect of HCP1 on protein levels of LC3-? and p62.4.5 HCP1 reduced the phosphorylation level of PEBP1(Ser153)in A549 cells without affecting its protein levels.In HEK293T cells,knockout of PEBP1 expression inhibited the effect of HCP1 on LC3-? protein levels.4.6 HCP1 induced chromatin condensation and PARP cleavage in A549 cells.4.7 HCP1 inhibited the growth of tumor and promoted tumor cell apoptosis in chicken embryo allantoic membrane.HCP1 did not affect the formation of chorioallantoic membrane blood vessels.CONCLUSIONS1.The coumarin pyrazoline derivative HCP1 inhibited the activity of HSP90 family members.HCP1 at low concentrations(1 ?M,2?M,5 ?M)selectively inhibited Grp94 activity by binding to site 3 of Grp94,while HCP1 at high concentration(10 ?M)also inhibited Hsp90?/? activity.2.Low-dose HCP1 inhibited oxLDL-induced HUVEC autophagy by inhibiting Grp94.Grp94 interacted with AMPK,HCP1 inhibited AMPK activity by inhibiting Grp94,and HCP1 suppressed autophagy by inhibiting AMPK-mTORC1 pathway.Therefore,Low-dose HCP1 suppressed oxLDL-induced HUVEC autophagy by inhibiting Grp94-AMPK-mTORC1 pathway.3.Low-dose HCP1 inhibited oxLDL-induced HUVEC apoptosis and cell viability reduction.4.HCP1 inhibited plaque endothelium autophagy,plaque cell apoptosis and increased plaque stability in apoE-/-mice.5.High-dose HCP1 inhibited A549 cell growth without causing cell necrosis.6.High-dose HCP1 induced autophagic flux injury and apoptosis in A549 cells.Phosphorylation of PEBP1(Ser153)may be involved in the damage of autophagic flux caused by HCP 1.