Designing Prodrug-type And Mitochondria-targeted Prooxidative Anticancer Agents By Exploiting Redox Achilles Heel Of Cancer Cells

Abnormal redox homeostasis has long been observed in cancer cells due to carcinogen stimulation,increased metabolic activity and mitochondrial dysfunction.Compared with normal cells,cancer cells requires higher level of reactive oxygen species（ROS）and redox active copper to maintain their malignant phenotypes;on the other hand,they are more dependent on higher concentrations of glutathione（GSH）to keep the ROS levels within the range that allows them to escape death under the ROS stress and maintain the homeostasis,thereby escaping apoptosis and realizing its proliferation,invasion and metastasis.Abnormal redox homeostasis of cancer cells including increased levels of GSH,ROS and copper along with mitochondrial dysfunction can be viewed as their redox Achilles heel,making them more vulnerable to further production of ROS using prooxidants,and thereby opening a window for developing prooxidative anticancer agents（PAAs）.However,it remains a challenge to design PAAs especially them with the unique ability to generate ROS selectively in cancer cells.Herein we developed natural product-inspired PAAs activated by redox Achilles heel of cancer cells and mitochondria-targeted PAAs,and even PAAs as concise theranostic molecules.We explored their ability to generate ROS selectively in cancer cells and the detailed prooxidative mechanisms by which they kill selectively cancer cells.The main contents are summarized as follows:（1）High levels of copper observed in cancer cells favours the idea of using copper inophores as PAAs to disrupt abnormal redox homeostasis of cancer cells.Herein we designed a3-hydroxyflavone-inspired copper pro-ionophore（PHF）as a potent PAA based on the GSH-mediated conversion of 2,4-dinitrobenzenesulfonates to enols.Mechanistic investigation reveals that it is capable of exploiting increased levels of GSH in cancer cells to in situ release an active ionophore,3-hydroxyflavone,inducing redox imbalance（copper accumulation,GSH depletion and ROS generation）and achieving highly selective killing of cancer cells upon specific transport of small amounts of Cu（Ⅱ）.To the best of our knowledge,it is the first example of Cu（Ⅱ）pro-ionophore type of PAA which hits the three birds with one stone（PHF）in terms of its ability to induce preferentially redox imbalance of cancer cells by copper accumulation,GSH depletion and ROS generation.（2）Inspired by the above work,we designed a naphthazarine-inspired copper pro-ionophore（Pro-Q2-2B）as a potent PAA based on the H₂O₂-mediated conversion of boronic ester to free phenol.Mechanistic investigation reveals that it is capable of exploiting increased levels of H₂O₂in cancer cells to in situ release an active copper ionophore,naphthazarine,inducing redox imbalance（copper accumulation,GSH depletion and ROS generation）and achieving highly selective killing of cancer cells.（3）We first used the GSH-mediated conversion of 2,4-dinitrobenzenesulfonates to phenols to protect a catechol moiety and developed stable pro-catechol-type diphenylpolyenes as small molecule-based prooxidative anticancer theranostic prodrugs.These molecules were synthesized via a modular route allowing creation of various pro-catechol-type diphenylpolyenes.As a typical representative,PDHH demonstrated three unique advantages:（1）capable of exploiting increased levels of GSH in cancer cells to in situ release a catechol moiety followed by its in situ oxidation to toxic o-quinone and ROS,leading to redox imbalance and final selective apoptosis of cancer cells;（2）permitting a turn-on fluorescent monitoring for its release,targeting mitochondria and therapeutic efficacy without the need of introducing additional fluorophores after its activation by GSH in cancer cells;（3）efficiently targeting mitochondria without the need of introducing additional mitochondria-directed groups.（4）Mitochondria function as a general cell death sensor by regulating the production of ROS to integrate many separate lethal signals.Thus,mitochondria are recognized as one of the most important targets for designing anticancer drugs.However,it is a great challenge for the target delivery of diagnostic agents to mitochondria of cancer cells with image-guided therapy and unique ability to generate ROS selectively in cancer cells.Herein we developed various catechol-type diphenylpolyenes as mitochondria-targeting PAAs by incorporation of a triphenylphosphonium（TPP）targeting group.These molecules were synthesized via a modular route allowing creation a variety of mitochondria-targeting diphenylpolyenes.As a typical representative,mito-DHH demonstrated four unique advantages:（1）the anticancer activity of mito-DHH is approximately 160 times more potent in A549 cells than its parent molecular DHH;（2）rapid accumulation of mito-DHH in mitochondria of cancer cells allows its ability to preferentially induce the collapse of membrane potential of cancer cells and interfere with their energy metabolism including inhibition of mitochondria OXPHOS,glycolysis and ATP generation;（3）mito-DHH could induce highly selective ROS(O₂^-·)accumulation of A549 over L02 cells to interfere with the redox homeostasis of cancer cells,leading to their selective killing;（4）mito-DHH allows a fluorescent monitoring for its mitochondria targeting and therapeutic effects.（5）Fluorescence probe has emerged as one of the most powerful techniques to monitor targets and biological processes in the context of a living system with high sensitivity,specificity,rapid detection,non-invasive,and in vivo applications.To facilitate mechanistic investigation on mitochondria-targeted PAAs by imaging mitochondrial cysteine（Cys）,we developed a novel fluorescent probe（CBMP）composed of phenyl methylpyridin-polyene（HBMP）as the fluorescence reporter,an acrylate moiety as the Cys response site,and a 1-methylpyridin-1-ium as the mitochondria-targeted carrier.This probe was designed by the strong“push-pull”effect of HBMP to effectively promote an ICT process.Our data reveal that CBMP works as a ratiometric fluorescence probe for sensing Cys with high selectivity and sensitivity（a fast response rate of 8min and a low detection limit of 82 nM）.More important,it was also successfully applied for two-photon imaging of mitochondrial Cys in living cells.