Discovery And Anticancer Study Of Small Molecules Inhibiting Thioredoxin Reductase

The thioredoxin system,which is composed of thioredoxin reductase(TrxR),thioredoxin(Trx)and NADPH,is a crucial antioxidant system in the maintenance of redox balance and regulation of redox-based signaling pathways.Accumulating evidence suggests that this system is a pivotal player closely related to many human diseases.The mammalian TrxR proteins,existing mainly as the cytosolic TrxR1 and mitochondrial TrxR2,are seleno-flavoenzymes containing the penultimate C-terminal selenocysteine(Sec)residue.TrxRs catalyze the transfer of electrons from NADPH to the active site of Trxs leading to the generation of reduced Trxs,and the reduced Trxs interact with downstream targets to regulate diverse redox-based intracellular responses involved in cell proliferation,differentiation and death.The higher thioredoxin levels usually exist in malignant cells than in normal cells,and thus targeting the Trx/TrxR system is thought to be an effective approach to impede tumor progression and metastasis.Therefore,the past years have witnessed continuing efforts on the development of small molecules targeting TrxR as potential therapeutic agents for cancer.Virtual screening is a strategy for the application of computer methods to select potential target compounds from chemical databases.The method of drug virtual screening is widely used to select potential active candidate compounds from a database of millions of drug-like molecules.Compared with the traditional processes,this method significantly reduce R&D costs and time.Structure-based virtual screening(SBVS)is applicable to the case where the receptor structure is known.The compound is docked with the target binding site selected in the previous stage,and the docking molecules are scored by predicting the binding pattern.The SBVS sorts are used as criteria for selecting potential molecules or combined with other evaluation methods.The experimentally evaluation of the selected compounds is performed to determine their biological activities on the molecular targets.This work successfully used the SBVS strategy to discover new TrxR inhibitors from hundreds of thousands of molecules in the natural product database,and fully demonstrates the efficiency and selectivity of the virtual screening strategy.We tested the toxic and enzyme inhibitory activities of the top-ranked 15 compounds,and the results showed that compounds 6,7,11 and 12 exhibited promising selective tumor cytotoxic activity.Meanwhile,the selected 15 compounds inhibited the extracellular pure TrxR enzyme and intracellular TrxR.Particularly,compounds 2,3,6,7,11 and 12 showed strong effects on the inhibition of TrxR.Based on the structural similarity of the target compounds,we analyzed the binding modes of compounds 2,7 and 12 using kinetic simulations.The binding mode analysis indicated that the non-covalent interactions between inhibitors and the TrxR active site promoted the covalent bonds formation between Sec498 in TrxR and the ?,?-unsaturated carbonyl in inhibitor.A series of non-covalent interactions with Lys29 and R351 on TrxR hinder the interactions between TrxR and Trx.The mechanism analysis showed that compound 7 inhibited the accumulation of oxidized Trx and increased ROS levels by inhibiting TrxR,which further promoted oxidative stress-mediated apoptosis in HeLa cells.Collectively,the virtual screening strategy to develop novel TrxR inhibitors could accelerate the discovery of TrxR inhibitors as potential therapeutics from small molecule databases.Based on the virtual screening research strategy and the dynamic simulation of the binding modes,we selected three natural products,xanthatin(XT),lipiferolide(LR)and cardamonin(CAR)to study the anticancer mechanism of targeting thioredoxin reductase.Among them,XT and LR were natural sesquiterpene lactones separated from Chinese herbal medicine Xanthium strumarium L.and Liriodendron,respectively,while CAR was a chalcone isolated from Alpinia katsumadai.These three compounds have been confirmed to show potential anticancer activity.In this paper,their anticancer mechanisms were studied.First,we demonstrated that XT,LR and CAR were effective in inhibiting extracellular and intracellular TrxR activity,and the Sec residue in the C-terminus of TrxR enzyme is the main inhibition target.Then,we analyzed the binding modes of TrxR with three compounds using the covalent docking method to verify the importance of Sec residue.Subsequently,the bioactivity experiments showed that the three natural small molecules could cause the production of intracellular reactive oxygen species,and further promoted the oxidative stress of tumor cells to induce apoptosis.In summary,our results explain for the first time the anticancer effects of three natural small molecule compounds targeting TrxR,which could provide data for the development of potential anticancer drugs targeting TrxR inhibitors.