Designing Mitochondria-targeting Prooxidative Anticancer Agents And Developing Fluorescent Probes For Monitoring Reactive Sulfur Species In Living Cells

Altered redox homeostasis and metabolic reprogramming are considered as two main characteristics of cancer.Compared with normal cells,cancer cells usually harbor increased levels of reactive oxygen species(ROS)to maintain their malignant phenotype,and are more dependent on increased levels of antioxidant enzymes and glutathione(GSH)to cope with the intrinsic oxidative stress.The altered redox homeostasis render cancer cells more vulnerable to further ROS generation,which has opened a window for developing the exogenous ROS-generating(prooxidative)agents kill cancer cells preferentially.However,it remains a challenge to design prooxidative anticancer agents with the unique ability to generate ROS in cancer cells efficiently and selectively.Moreover,cancer cells undergo metabolic reprogramming(aerobic glycolysis)to support their increased needs for ATP generation and macromolecule biosynthesis,which becomes an attractive target for cancer therapy.However,precise and effective intervention in cancer metabolic reprogramming is still a key question,due to metabolic heterogeneity and plasticity of cancer cells.Mitochondria,being a vital organelle of eukaryotic organisms,play an important role in a wealth of cellular processes such as energy supply and ROS generation.According to the significant difference in structure and function of mitochondria between cancer and normal cells,we designed natural product-inspired prooxidative anticancer agents based on a mitochondria-targeting strategy.These molecules will be used to probe their ability to selectively kill cancer cells by promoting ROS production,which inevitably invites the following questions of(1)whether they can generate ROS in cancer cells efficiently and selectively through targeting mitochondria,and(2)if so,whether the resulting ROS are capable of mediating preferentially energy crisis of cancer cells in a dual inhibition fashion against both oxidative phosphorylation and glycolysis.The major contents are summarized as follows:(1)We designed the mitochondrial-targeting compounds MitoCur-1 and Mito-XN by incorporating the triphenylphosphonium moiety with natural curcumin and xanthohumol as the leads,respectively.Mechanistic investigation suggests that benefited from their Michael acceptor units,they can produce ROS with high efficacy and selectivity in cancer cells over normal cells via inhibition against mitochondrial antioxidative thioredoxin reductase(TrxR);more importantly,the ROS generation preferentially induces energy crisis and death of cancer cells in a dual inhibition fashion against both oxidative phosphorylation and glycolysis.These results highlight a general strategy for effective intervention in cancer metabolic reprogramming,that is,inhibiting mitochondrial TrxR to generate ROS in cancer cells efficiently and selectively by Michael acceptor-type molecules.Moreover,MitoCur-1 was identified to be superior to the regular chemotherapeutic agents such as 5-fluorouracil,gemcitabine and doxorubicin,in killing preferentially human hepatoma Hep G2 cells over human normal liver L02 cells,and exhibited high antitumor efficacy in vivo,BALB/c nude mice bearing Hep G2 tumors.(2)Angiogenesis has been recognized as a key step for tumor metastasis because it delivers oxygen and nutrients for growing tumor,therby representing a promising target for cancer therapy.However,relatively few researches focus on inhibiting angiogenesis from a prooxidative point of view.Herein we designed xanthohumol and its analogues to probe their structural basis and mechanism in generating ROS and inhibiting angiogenesis.Based on the endothelial cell migration,chick chorioallantoic membrane and fluorescence imaging assays,we demonstrated that compound XN-2 can covalently modify TrxR and deplete GSH by virtue of its Michael acceptor unit,thereby promoting accumulation of ROS and inhibiting angiogenesis.The above results give useful information for designing Michael acceptor-based prooxidative angiogenesis inhibitors.Reactive sulfur species(RSS)include sulfur dioxide(SO₂)and various biothiols,such as hydrogen sulfide(H₂S),GSH,cysteine(Cys),and homocysteine(Hcy).These RSS are closely related to each other through complicated symbiotic networks and play crucial roles in redox homeostasis,signal conduction and metabolic regulation.Thus,accurate and rapid detection of intracellular RSS is imperative to elucidate its role in physiological and pathological processes.Nowadays,the fluorescent detection method has attracted much attention owing to its unique properties,such as nondestructive detection,outstanding sensitivity,strong visibility and excellent selectivity.Considering that RSS is a major participant in redox homeostasis,rationally designing the fluorescent probes to discriminate them should be promising tools for facilitating the pro-oxidative anticancer mechanistic investigation.Accordingly,we designed two fluorescent probes for tracking cytoplasmic and mitochondrial RSS in living cells.The major contents are summarized as follows:(3)GSH,the most abundant intracellular biothiol,works as a cornerstone of maintaining redox homeostasis.However,it is still a challenge for effectively discriminating GSH from other biothiols including Cys and Hcy,owing to their similar chemical properties.Herein,by modifying2-(2?-hydroxyphenyl)benzothiazole(HBT)scaffold,we constructed an excited-state intramolecular proton transfer(ESIPT)-based fluorescent probe BTFMD for selectively detecting GSH,where 2,4-dinitrobenzenesulfonyl group functions as the trigger for its rapid cleavage to BTFM-OH by GSH,Cys and Hcy.To reduce the interference from Cys and Hcy,we incorporated an aldehyde moiety that could further cyclize with them,thereby quenching green fluorescence of BTFM-OH.The probe is characterized by good selectivity,large Stokes shift,fast response rate and easy preparation.Subsequently,the probe was successfully applied to explore the role of GSH in the development of cancer and Parkinson's disease.Furthermore,the probe showed the satisfactory performance of fluorescence imaging in zebrafish.(4)SO₂ and various biothiols such as H₂S,GSH,Cys,and Hcy,are regarded as essential molecules of RSS,and tightly associated with various clinical diseases.Currently,it remains a challenge to develop a single fluorescent probe to differentiate RSS simultaneously,due to the fact that these RSS harbor similarity in chemical properties,such as nucleophilicity.Herein,we subtly integrated a hybrid of benzopyrylium with coumarin(NIR-OH)and a nitro benzoxadiazole(NBD)into a single molecule through an ether bond to design a new multi-signal fluorescent probe NIR-NBD.The probe NIR-NBD features three different types of electrophilic sites with the activity order of 1>2>3,in the part of coumarin,benzopyrylium and NBD,respectively.NIR-NBD is characterized of simultaneously discriminating Cys/Hcy/H₂S,GSH,and SO₂ from each other in a fashion of three sets of distinct signals:red and green for Cys/Hcy/H₂S,red for GSH,and green for SO₂.The probe manifested a favorable comprehensive performance including fast response,easy preparation,good water solubility and selectivity.Most notably,the probe displayed excellent selectivity,outstanding sensitivity(detection limit=3.5 n M)and ultrafast response(within 4 min)toward SO₂.Finally,the probe was successfully applied for discriminative imaging of Cys/Hcy/H₂S,GSH,and SO₂ in living cells,and visualization of the endogenous generation of SO₂ and investigation on the role of mitochondrial biothiols during apoptosis process.