Abasic (AP) sites are one of the most common DNA lesions in cells. Aldehyde-reactive alkoxyamines capture AP sites and block the activity of APE1, the enzyme responsible for initiating their repair. Blocking the APE1 repair of AP sites leads to cell death, and it is an actively investigated approach for treating cancer. However, unselective AP site capture in different cells produces side effects and limits the application of alkoxyamines in chemotherapy. Herein we take advantage of the higher glutathione (GSH) concentration in cancer cells over normal cells to develop GSH-inducible agents that selectively kill cancer cells. 2,4-Dinitrobenzenesulfonamide caged coumarin-based alkoxyamines 1 and 2 are selectively revealed by GSH to release SO2 and fluorescent coumarin-based alkoxyamines 3 and 4 that trap AP sites in cells. GSH-directed AP site trapping and SO2 release result in selective cytotoxicity (defined as IC50WI38/IC50H1299) against H1299 lung cancer cells over normal WI38 lung cells, ranging from 1.8 to 2.8 for 1 and 2. The alkylating agent methylmethanesulfonate (MMS) promotes the formation of AP sites in cells and enhances the cytotoxicity of agent 1 in a dose-dependent way. Moreover, the comet assay and γH2AX assay suggest that AP adducts form a highly toxic DNA interstrand cross-link (ICL) upon photolysis, leading to further cell death. DNA flow cytometric analysis showed that 1 promoted cell apoptosis in the early stage and induced G2/M phase cell-cycle arrest. The 2,4-dinitrobenzenesulfonamide-caged alkoxyamines exhibited selective antitumor activity and photocytotoxicity in cancer cells, illuminating their potential as GSH-directed chemotherapeutic agents.
DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions, and induce cell death by blocking DNA strand separation. Most ICL agents aiming to kill cancer cells, also generate adverse side effects to normal cells. H 2 O 2 -inducible DNA ICL agents are highly selective for targeting cancer cells, as the concentration of H 2 O 2 is higher in cancer cells than normal cells. Previous studies have focused on arylboronate-based precursors, reacting with H 2 O 2 to generate reactive quinone methides (QMs) crosslinking DNA. Here we explore phenyl selenide-based precursors 1-3 as H 2 O 2 -inducible DNA ICL agents. The precursors 1-3 can be activated by H 2 O 2 to generate the good benzylic leaving group and promote production of reactive QMs to crosslink DNA. Moreover, the DNA cross-linking ability is enhanced by the introduction of substituents in the paraposition of the phenolic hydroxyl group. From the substituents explored (H, OMe, F), the introduction of electron donating group (OMe) shows a pronounced elevating effect. Further mechanistic studies at the molecular and DNA levels confirm alkylation sites located mainly at dAs, dCs and dGs in DNA. Additionally, cellular experiments reveal that agents 1-3 exhibit higher cytotoxicity toward H1299 human lung cancer cells compared to clinically used drugs, by inducing cellular DNA damage, apoptosis and G0/G1 cell cycle arrest. This study provides a strategy to develop H 2 O 2 -inducible DNA interstrand cross-linkers.
Proteolysis‐targeting chimeras (PROTACs) provide a powerful technique to degrade targeted proteins utilizing the cellular ubiquitin‐proteasome system. The major concern is the host toxicity resulting from their poor selectivity. Inducible PROTACs responding to exogenous stimulus, such as light, improve their specificity, but it is difficult for photo‐activation in deep tissues. Herein, we develop H2O2‐inducible PROTAC precursors 2/5, which can be activated by endogenous H2O2 in cancer cells to release the active PROTACs 1/4 to effectively degrade targeted proteins. This results in the intended cytotoxicity towards cancer cells while targeted protein in normal cells remains almost unaffected. The higher Bromodomain‐containing protein 4 (BRD4) degradation activity and cytotoxicity of 2 towards cancer cells is mainly due to the higher endogenous concentration of H2O2 in cancer cells (A549 and H1299), characterized by H2O2‐responsive fluorescence probe 3. Western blot assays and cytotoxicity experiments demonstrate that 2 degrades BRD4 more effectively and is more cytotoxic in H2O2‐rich cancer cells than in H2O2‐deficient normal cells. This method is also extended to estrogen receptor (ER)‐PROTAC precursor 5, showing H2O2‐dependent ER degradation ability. Thus, we establish a novel strategy to induce targeted protein degradation in a H2O2‐dependent way, which has the potential to improve the selectivity of PROTACs.
Biothiols play essential roles in balancing redox state and modulating cellular functions. Fluorescent probes for monitoring/labelling biothiols often suffer from slow reaction rates, strong background fluorescence and cytotoxic byproducts release....
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