Application Of Cyclic Disulfide/Diselenide Scaffold In Constructing Small Molecules To Target Cellular Redox-Regulating Systems

Thioredoxin reductase?TrxR?/thioredoxin?Trx?and glutathione reductase?GR?/glutathione?GSH?are two major antioxidant systems which based on thiol/disulfide-like exchange reactions.These two systems synergize to keep cellular protein thiol/disulfide and redox in balance.Mounting evidence indicated that TrxR and GSH are overexpressed in tumor cells and the upregulation of TrxR and GSH are essential for maintaining tumor phenotypes.In this thesis,we first synthesized a series of fluorescent probes based on cyclic disulfides/diselenides and systematically studied the selectivity of these probes to TrxR and GSH.According to the redox properties of the cyclic disulfides/diselenides,we developed a superfast and highly specific fluorogenic probe of TrxR Fast-TRFS.We then utilized the 5-membered cyclic disulfide to develop the first TrxR-activated prodrug S-Gem.By the utilization of the cyclic diselenides,a GSH-activated prodrug Se-Gem was also synthesized.As asparagus acid?AA?,a cyclic disulfide-containing compound,is a natural product derived from foods.We then evaluated the neuroprotective properties of this compound.The results are summarized as follows:Chapter 1.The role of modulation of antioxidant enzyme systems in the treatment of diseases and the methods of selective evaluation of TrxR activity were summarized firstly.Then the cellular antioxidant proteins based on thiol/selenol-disulfide exchange reactions and the applications of cyclic disulfides/diselenides in designing probes or drugs were reviewed.Chapter 2.Fast-TRFS,a superfast and specific fluorescent probe of TrxR with a novel sensing mechanism,has been discovered.The response rate was improved by dissecting the structural determinants of the cyclization-driven release process,and the high specificity was achieved by exploring the selectivity of cyclic disulfides/diselenides reduction by TrxR.The favorable properties of Fast-TRFS enabled the development of a high-throughput screening assay to discover TrxR inhibitors by using crude tissue extracts as a TrxR source,and dozens of new natural TrxR inhibitors were identified.Chapter 3.Conjugation of the 1,2-dithiolane moiety with the anticancer drug gemcitabine?Gem?via a carbamate linker generates the prodrug S-Gem,which is specifically activated by TrxRs to liberate the parent drug Gem.S-Gem shows less cytotoxicity compared to Gem,but exhibits TrxR-dependent cytotoxicity.As overexpression of TrxR is frequently found in different types of tumors,the TrxR-dependent prodrugs are promising for further development as cancer chemotherapeutic agents.Chapter 4.Conjugation of a redox-responsive scaffold 1,2-diselenolan-4-ol to the anticancer drug Gem to obtain a prodrug Se-Gem.Se-Gem is effectively activated by GSH,the most abundant cellular thiols,through thiol/diselenide exchange reaction to release the the parent drug Gem and an alkyl selenol-containing compound,which is rapidly oxidized by the ambient molecular oxygen to generate superoxide anions.Further studies reveal that the liberated selenol-containing compound can act as a redox cycler which promotes continuous depletion of reduced GSH and total thiols,accumulation of reactive oxygen species?ROS?and induction of apoptosis in cancer cells.As cancer cells are more prone to developing drug resistance,the synthesis of Se-Gem provides a promising therapeutic agent and a novel drug delivery system to improve therapeutic activity and selectivity of the parent drug in cancer therapy.Chapter 5.The neuroprotective effect of asparagusic acid?1,2-dithiolane-4-carboxylic acid,AA?was evaluated.We found that AA could protect PC12 cells against H₂O₂-and 6-OHDA-induced oxidative damage.Mechanistic studies indicated that AA could activate the Keap1-Nrf2-ARE signal pathway and induce the expression of a battery of cytoprotective genes as well as the activities of the corresponding gene products.Chapter 6.We summed up the whole thesis,and gave study prospect of further applications of cyclic disulfides/diselenides in drug design.