Designing Natural Product-inspired Cancer Chemopreventive And Neuroprotective Agents By Targeting Nrf2/ARE Signaling Pathway

Oxidative stress might play a causative role in cancer and neurodegenerative diseases.Activating Nrf2/ARE signaling pathway represents one of the most important cellular defense mechanisms against oxidative stress,and is also a proven means of developing indirect antioxidants,cancer chemopreventive and neuroprotective agents.The signaling pathway,however,is like a double-edged sword and has double effects.Constitutive Nrf2 activation in many tumors enhances cell survival and resistance to anticancer drugs.Therefore,there is no denying the fact that designing and discovering both Nrf2 activators and inhibitors have important significance for developing anti-cancer agents.In this thesis,based on highly reactive and nucleophilic cysteine residues on Keap1 protein,the structurally diverse molecules inspired by resveratrol,curcumin and piperlongumine were selected and designed form the point view on oxidation potential,electrophilicity and so on to find more active Nrf2 activators than the parent molecules.These compounds were chosen to probe their abilities,structural determinants and adaptive protection mechanism to act as Nrf2 activators in the two established models of oxidative stress in HepG2 and PC-12 cells induced by tert-butyl hydroperoxide（t-BHP）and6-hydroxydopamine（6-OHDA）,respectively.Additionally,we identified natural isoliquiritigenin as an Nrf2 inhibitor to sensitize drug-resistant cells to cisplatin.The main achievements and innovations are summarized next:（1）Catechol moieties are commonly present in dietary natural products that exert cancer chemopreventive activity.While the oxidative conversion of catechols into their corresponding o-quinones is generally considered to contribute to their cancer chemopreventive effects,the mechanism of the intracellular conversion has not been fully elucidated.Among resveratrol and its hydroxylated analogs examined,only 3,4-dihydroxy-trans-stilbene（3,4-DHS）exerted cytoprotective effects against t-BHP-induced death of HepG2 cells.This resveratrol analog activated the Nrf2 pathway through stimulating phosphorylation of Akt and inducing Keap1modification,thereby resulting in its nuclear translocation and subsequent transcriptional induction of phase II detoxifying enzymes.Its cytoprotective effect through Nrf2 activation was largely abrogated by pretreatment of cells with DTT,a sulfhydryl-containing nucleophile,and neocuproine,a specific chelating agent for copper ions.We identified 3,4-DHS as a novel Nrf2activator that is converted intracellularly into its corresponding o-quinone electrophile by copper ions.The copper-mediated oxidation was required for the Nrf2 activation,subsequent transcriptional induction of phase II detoxifying enzymes and ultimately for cytoprotection.The findings demonstrate a previously underrecognized role for intracellular copper ions in the cancer chemopreventive effects of catechol-containing dietary natural products.We believe that the above findings may have broad chemical and biological implications for elucidating the importance of a catechol moiety in cancer chemoprevention,understanding why dietary natural products with this moiety tend to show cancer chemopreventive activity,and designing polyphenol-inspired cancer chemopreventive agents.（2）Considering that the copper-mediated oxidation of 3,4-DHS is a key step for its Nrf2activation,form the point view on decreasing its oxidation potential as well as increasing its lipophilicity and copper ion-binding ability,we designed its analog 3,4-DHD by retaining its catechol moiety and elongating its conjugated links,and found that 3,4-DHD was a more potent Nrf2 activator and neuroprotective agent in 6-OHDA-induced oxidative injury of PC12 cells.（3）Based on the electrophilicity-and stability-increasing strategy,we designed a double o-trifluoromethyl substituted diarylpentadienone（A1）with curcumin as the lead compound.It was found that compared with curcumin,this molecule displayed significantly increased Nrf2-activating and neuroprotective activities via Michael acceptor-dependent mechanisms by which it stimulated phosphorylation of Akt and covalently modified Keap1 protein,thereby resulting in nuclear translocation of Nrf2,decreased ubiquitination of Nrf2 and subsequent transcriptional induction of phase II detoxifying enzymes.（4）Based on the electrophilicity-,metabolism stability-and lipophilicity-increasing strategy,we designed a piperlongumine analogue PLCl-4CF₃ by placing a trifluoromethyl group on its aromatic ring in the para position and introducing an electron-withdrawingα-Cl on its lactam ring.We identified PLCl-4CF₃ as a novel piperlongumine-directed Nrf2 activator to exhibit significant cytoprotective effects against 6-OHDA-induced oxidative injury of PC12 cells.Notably,its Nrf2-activating mechanisms were analogous to those of A1,but phosphorylation of Akt was not responsible for the activation.（5）We found that natural isoliquiritigenin could sensitize drug-resistant human ovarian cancer SKOV3/DDP cells to cisplatin by inhibiting Nrf2/ARE signaling pathway.Mechanistic investigation revealed that at least two posttranslational modifications,that is,inhibiting phosphorylation of Akt and intensifying ubiquitination of Nrf2 were related with the process.