Glutathione-responsive PROTAC for targeted degradation of ERα in breast cancer cells.

ERα (estrogen receptor-α)-targeting PROTACs (PROteolysis TArgeting Chimeras) have emerged as a novel and promising modality for breast cancer therapeutics. However, ERα PROTACs-induced degradation in normal tissues raises concerns about potential off-tissue toxicity. Tumor microenvironment-responsive strategy provides potential for specific control of the PROTAC's on-target degradation activity. The glutathione (GSH) level has been reported to be significantly increased in tumor cells. Here, we designed a GSH-responsive ERα PROTAC, which is generated by conjugating an o-nitrobenzenesulfonyl group to the hydroxyl group of VHL-based ERα PROTAC through a nucleophilic substitution reaction. The o-nitrobenzenesulfonyl group as a protecting group blocks the bioactivity of ERα PROTAC (ER-P1), and that can be specifically recognized and removed by highly abundant GSH in cancer cells. Consequently, the GSH-responsive ERα PROTAC (GSH-ER-P1) exhibits significantly enhanced degradation of ERα in cancer cells compared to that in normal cells, leading to a remarkable inhibition of breast cancer cell proliferation and less toxic effects on normal cells. This study provides a potentially valuable strategy for breast cancer treatment using tumor microenvironment-responsive PROTACs.

Dual-responsive probe and DNA interstrand crosslink precursor target the unique redox status of cancer cells.

Elevated GSH and H2O2 in cancer cells is sometimes doubted due to their contrary reactivities. Here, we construct a dual-responsive fluorescent probe to confirm the conclusion, and employ this to exploit a redox-inducible DNA interstrand crosslink (ICL) precursor. It crosslinks DNA upon activation by GSH and H2O2, affording an alternative dual-responsive strategy.

A novel fluorescent dye selectively images and kills cancer stem cells by targeting mitochondria: Evidence from a cell line­based zebrafish xenograft model.

Numerous agents such as near-infrared dyes that are characterized by specialized cancer imaging and cytotoxicity effects have key roles in cancer diagnosis and therapy via molecularly targeting special biological tissues, organelles and processes. In the present study, a novel fluorescent compound was demonstrated to inhibit cancer cell proliferation in a zebrafish model with slight in vivo toxicity. Further studies demonstrated selective staining of cancer cells and even putative cancer stem cells via accumulation of the dye in the mitochondria of cancer cells, compared with normal cells. Moreover, this compound was also used to image cancer cells in vivo using a zebrafish model. The compound displayed no apparent toxicity to the host animal. Overall, the data indicated that this compound was worthy of further evaluation due to its low toxicity and selective cancer cell imaging and killing effects. It could be a useful tool in cancer research.

Selective degradation of BRD4 suppresses lung cancer cell proliferation using GSH-responsive PROTAC precursors.

BRD4,as a transcriptional and epigenetic regulator to mediate cellular functions, plays an important role in cancer development.Targeting BRD4 with conventional inhibitors in cancer therapy requires high doses, which often leads to off-target and adverse effects. BRD4-targeted proteolysis-targeting chimeras (PROTACs) can catalytically degrade BRD4 utilizing the endogenous proteasome system, and exhibit promising anti-tumor activity. However, most of the developed PROTACs are non-cancer specific and relatively toxic towards normal cells, limiting their practical applications in cancer treatment. By taking advantage of higher glutathione (GSH) levels in cancer cells than that in normal cells, we developed several GSH-responsive PROTAC precursors 1a-c via the attachment of a GSH-trigger unit on the hydroxyl group of the VHL (von Hippel-Lindau) ligand for the recruitment of E3 ligase. Among the precursors, 1a can be efficiently activated by the innately higher concentrations of GSH in lung cancer cells (A549 and H1299) to release active PROTAC 1, degrading intracellular BRD4 and resulting in cytotoxicity, which is confirmed by mechanistic investigation. On the other hand, 1a cannot be efficiently triggered in normal lung cells (WI38 and HULEC-5a) containing lower levels of GSH, therefore reducing the adverse effects on normal cells. This work provides an alternative proof of concept approach for developing stimuli-responsive PROTAC precursors, and affords a novel insight to improve the selectivity and minimize the adverse effects of current PROTACs, hence enhancing their clinical potential.

Hydrogen Peroxide-Inducible PROTACs for Targeted Protein Degradation in Cancer Cells.

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 H2 O2 -inducible PROTAC precursors 2/5, which can be activated by endogenous H2 O2 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 H2 O2 in cancer cells (A549 and H1299), characterized by H2 O2 -responsive fluorescence probe 3. Western blot assays and cytotoxicity experiments demonstrate that 2 degrades BRD4 more effectively and is more cytotoxic in H2 O2 -rich cancer cells than in H2 O2 -deficient normal cells. This method is also extended to estrogen receptor (ER)-PROTAC precursor 5, showing H2 O2 -dependent ER degradation ability. Thus, we establish a novel strategy to induce targeted protein degradation in a H2 O2 -dependent way, which has the potential to improve the selectivity of PROTACs.

Light and hydrogen peroxide dual-responsive DNA interstrand crosslink precursors with potent cytotoxicity.

Arylboronic acid/esters and phenyl selenides-based quinone methide (QM) precursors were reported to induce DNA interstrand crosslink (ICL) formation upon reaction with the inherently high concentrations of H2O2 in cancer cells. However, some normal cells (such as macrophages) also contain high-levels of H2O2, which may interfere with precursors' selectivity. In order to enhance the spatiotemporal specificity by the photolysis, we developed photo- and H2O2- dual-responsive DNA ICL precursors 1-3, bearing a photo-responsive coumarin moiety and a H2O2 inducible phenyl selenide group. Precursors 1-3 are efficiently activated by photoirradiation and H2O2 to generate reactive QMs crosslinking DNA. Moreover, the reactivity of precursors can be modulated by the introduction of aromatic substituents (OMe, F), and the electron donating group (OMe) displays a more pronounced promoting effect on DNA ICL formation. A subsequent piperidine heat stability study confirmed that the formed QMs primarily alkylate dAs, dGs and dCs in DNA. Furthermore, 1-3 inhibit lung cancer cell (H1299) growth by inducing DNA damage and producing toxic reactive oxygen species (ROS) upon photolysis of released coumarin. This study illustrates the potent cytotoxicity achieved by novel photo/H2O2 dual-responsive QM precursors 1-3, affording a novel strategy for the development of inducible DNA interstrand cross-linkers.

Rapid GSH detection and versatile peptide/protein labelling to track cell penetration using coumarin-based probes.

Biothiols play essential roles in balancing the redox state and modulating cellular functions. Fluorescent probes for monitoring/labelling biothiols often suffer from slow reaction rates, strong background fluorescence and cytotoxic byproduct release. Thus, developing facile and versatile probes to overcome the challenges is still in high demand. Here, we report four coumarin-maleimides as fast responding and fluorogenic probes to detect GSH or label peptides/proteins. The probes quantitatively and selectively react with GSH via Michael addition within 1-2 min, achieving an 11-196-fold increase in fluorescence quantum yield via blockage of the photoinduced electron transfer (PET) process. Optimized probe 4 is applied for the detection of GSH in vitro (A549 cells) and in vivo (zebrafish embryos). Taking advantage of the fast Michael addition between the maleimide moiety and the sulfhydryl group, we expand the application of our method for fluorescent labelling of peptides/proteins and for tracking their cellular uptake process. The labelling strategy works for both Cys-bearing and Cys-free proteins after the introduction of a sulfhydryl group using Traut's reagent. Fluorescence assay reveals that the TAT-peptide can efficiently enter cells, but H3 protein, part of nucleosomes, prefers to bind on the cell membrane by electrostatic interactions, shedding light on the cellular uptake activity of nucleosomes and affording a potential membrane staining strategy. Overall, our study illustrates the broad potential of coumarin-maleimide based dual-functional probes for GSH detection and versatile protein labelling in biochemical research.

Selective Antitumor Activity and Photocytotoxicity of Glutathione-Activated Abasic Site Trapping Agents.

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.

Phenyl Selenide-Based Precursors as Hydrogen Peroxide Inducible DNA Interstrand Cross-Linkers.

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. H2 O2 -inducible DNA ICL agents are highly selective for targeting cancer cells, as the concentration of H2 O2 is higher in cancer cells than normal cells. Previous studies have focused on arylboronate-based precursors, reacting with H2 O2 to generate reactive quinone methides (QMs) crosslinking DNA. Here we explore phenyl selenide-based precursors 1-3 as H2 O2 -inducible DNA ICL agents. The precursors 1-3 can be activated by H2 O2 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 para-position 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 H2 O2 -inducible DNA interstrand cross-linkers.

A novel double target fluorescence probe for Al3+/Mg2+ detection with distinctively different responses and its applications in cell imaging.

The metal cations, Al3+ and Mg2+, could affect human health and cell biological processes. Their fast and selective detection using one probe remains a challenge. A novel fluorescence probe, N'-((1-hydroxynaphthalen-2-yl)methylene)isoquinoline-3-carbohydrazide (NHMI), was developed for selectively monitoring Al3+ and Mg2+. The probe NHMI showed a distinctive "turn-on" fluorescence signal towards Al3+ and Mg2+ (cyan for Al3+ with 2556-folds enhancement and yellow for Mg2+ with 88-folds enhancement), which is quite distinct from other metal cations and allows for naked-eye detection. This interesting response was attributed to the influence of PET, ESIPT process and CHEF effect, when Al3+ or Mg2+ chelated with NHMI. Furthermore, the fluorescence titration experiments manifested that the detection limit of probe NHMI for Al3+/Mg2+ was as low as 1.20 × 10-8 M and 7.69 × 10-8 M, respectively. The formed complexes NHMI-Al3+ and NHMI-Mg2+ were analyzed by Job's plot, ESI-MS, 1H NMR and FT-IR. The coordination pockets and fluorescence mechanisms of two metal complexes were explored by density functional theory calculation. Moreover, NHMI showed low cytotoxicity and good cell permeability. Fluorescence bioimaging of Al3+/Mg2+ in MCF-7 cells with NHMI indicated its potential application in biological diagnostic analysis.

Discovery and Optimization of Novel Hydrogen Peroxide Activated Aromatic Nitrogen Mustard Derivatives as Highly Potent Anticancer Agents.

We describe several new aromatic nitrogen mustards with various aromatic substituents and boronic esters that can be activated with H2O2 to efficiently cross-link DNA. In vitro studies demonstrated the anticancer potential of these compounds at lower concentrations than those of other clinically used chemotherapeutics, such as melphalan and chlorambucil. In particular, compound 10, bearing an amino acid ester chain, is selectively cytotoxic toward breast cancer and leukemia cells that have inherently high levels of reactive oxygen species. Importantly, 10 was 10-14-fold more efficacious than melphalan and chlorambucil for triple-negative breast-cancer (TNBC) cells. Similarly, 10 is more toxic toward primary chronic-lymphocytic-leukemia cells than either chlorambucil or the lead compound, 9. The introduction of an amino acid side chain improved the solubility and permeability of 10. Furthermore, 10 inhibited the growth of TNBC tumors in xenografted mice without obvious signs of general toxicity, making this compound an ideal drug candidate for clinical development.

Hydrogen peroxide activated quinone methide precursors with enhanced DNA cross-linking capability and cytotoxicity towards cancer cells.

Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development.

Template-directed fluorogenic oligonucleotide ligation using "click" chemistry: detection of single nucleotide polymorphism in the human p53 tumor suppressor gene.

A novel nonfluorescent alkyne-modified coumarin phosphoramidite was synthesized and successfully incorporated into oligonucleotides, which were then used in highly efficient DNA interstrand cross-linking and ligation reactions via "click" chemistry. The template-directed fluorogenic ligation "click" chemistry reaction was used for single nucleotide polymorphism analysis, where the target DNA catalyzes the ligation of two nonfluorescent probes to generate a fluorescent product. The upstream oligonucleotide probe is a nonfluorescent alkyne-modified coumarin and the downstream probe is an azide-modified oligonucleotide. When bound to a fully complementary template, the oligonucleotides ligated to produce a fluorescent product with a fluorophore at the ligation point. Wild-type and mutant p53 alleles were used to demonstrate that template-directed fluorogenic oligonucleotide ligation is sequence-specific and is capable of single nucleotide discrimination under mild conditions, even without the removal of unreacted probes.