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Interlocked DNA nanostructures perform programmable movements in nanoscales such as sliding, contraction, and expansion. However, utilizing nanoscaled interlocked movements to regulate the functions of larger length scaled matrix and developing their applications has not yet been reported. Herein we describe the assembly of DNA-based daisy chain rotaxane nanostructure (DNA-DCR) composed of two hollow DNA nanostructures as macrocycles, two interlocked axles and two triangular prism-shaped DNA structures as stoppers, in which three mechanical states─fixed extended state (FES), sliding state (SS), and fixed contracted state (FCS)─are characterized by using toehold-mediated strand displacement reaction (SDR). The DNA-DCRs are further used as nanocomposites and introduced into hydrogel matrix to produce interlocked hydrogels, which shows modulable stiffness by elongating the interlocked axles to regulate the hydrogel swelling with hybridization chain reaction (HCR) treatment. Then the DCR-hydrogels are employed as dynamic biointerfaces for human mesenchymal stem cells (hMSCs) adhesion studies. First, hMSCs showed lower cell density on bare DCR-hydrogel treated with HCR-initiated swelling for stiffness decreasing. Second, the cell adhesion ligand (RGD) modified DNA-DCRs are constructed for hydrogel functionalization. DCR

Diabetic patients with type 1 or advanced type 2 stages need timely and precise insulin injection to regulate the daily blood glucose levels (BGLs). Otherwise, risks of serious or even deadly diabetes-associated complications occur. To achieve prolonged glucose regulation and low hypoglycemia risks, a novel on-demand glucose-responsive glycopolymer system was constructed for insulin delivery, which was self-assembled into nanoparticles by dynamic covalent bonds between two polymers: fluorophenylboronic acid-grafted polymer (poly-F) and polyol polymer (poly-G). Insulin was loaded during the assembly process. The nanoparticles showed excellent glucose responsiveness

Diabetes is a metabolic disease that is affecting an ever-increasing number of people worldwide, resulting in increased burdens on healthcare systems and societies. Constant monitoring of blood glucose levels is required to prevent serious or even deadly complications. One major challenge of diabetes management is the simple and timely administration of insulin to facilitate consistent blood glucose regulation and reduce the incidence of hypoglycemia. With this research, we construct an insulin delivery system, the delivery system is comprised of phenylboronic acid based fluorescent probes, which is used as glucose responsive linkers, mesoporous silica nanoparticles providing an insulin reservoir, and zinc oxide nanoparticles used as gate keepers. The system with glucose sensitive responsive linker exhibits controlled release of insulin under high glucose concentrations, providing prolonged blood glucose regulation and no risks of hypoglycemia. Furthermore, the system is combined with a hyaluronic-acid based microneedle patch, which exhibit efficient skin penetration for transdermal delivery. With our system, the nanoparticles provide outstanding in vivo glucose regulation when administrated by subcutaneous injection or via transdermal microneedle patch. We anticipate that our biocompatible smart glucose responsive microneedle patch (SGRM patch) will facilitate the development of clinically useful systems.

Multi-modal combination therapy has attracted great attention, owing to the unsatisfactory therapeutic efficacy of conventional chemotherapy. Mesoporous silica-coated gold nanorods possess great potential in photothermal therapy and drug delivery. In this work, we fabricate a dual-responsive nanohybrid for combination treatment of the malignant tumor. In this system, gold nanorods are coated with the degradable mesoporous silica, and the chemotherapy drug doxorubicin (DOX) and photosensitizer (IR820) are co-loaded inside the pores of the silica. The encapsulation of hyaluronic acid (HA) endow the nanohybrids with mammary carcinoma targeting ability and better biocompatibility, owning to CD44+ receptor overexpressed in some cancer cells. As-prepared nanohybrids exhibit high responsiveness to a high glutathione (GSH) level and degrade rapidly in the presence of hyaluronidase (HAase) and GSH after endocytosis by 4T1 cells, allowing the efficient release of loaded DOX and IR 820 in tumor sites. Interestingly, near-infrared (NIR) laser not only triggers the generation of reactive oxygen species, but also remarkable photothermal efficacy originating from GNRs. Therefore, upon the irradiation of 808 nm NIR light, the combinatorial photodynamic, photothermal and chemotherapy is achieved, accordingly leading to a highly efficient antitumor outcome in vitro and in vivo. This strategy provides an ideal approach to constructing multimodal cancer therapy system. STATEMENT OF SIGNIFICANCE: • Dual-responsive nanohybrids for combinatorial therapy of breast cancer. • The nanohybrids exhibit both HAase and GSH stimuli-responsive behavior. • The nanohybrids exhibit light-activated PDT/PTT/chemotherapy. • The nanohybrids show good biosafety for potential clinical application.

Nitric oxide donors (NODs) are indispensable in biological research and disease treatment. NODs had been utilized to treat cardiovascular diseases in clinic and many others are under trial. Thiols are typically required for these donors to release NO. Yet, their mechanism is complex and often lead to resistance. Herein, we reported that N-nitrosated electron-deficient dyes are capable of NO release with one-electron reduction. A fluorophore is generated simultaneously, whose fluorescence is harnessed to monitor the profile of NO release. Through electrochemical and spectral studies, NOD f3 was found to exhibit good biocompatibility and high reduction efficiency and its potentials in cell-protection in oxygen and glucose deprivation (OGD) models were showcased with endothelial cells. This work aims at offering a new approach to design reduction-triggered NOD, which have therapeutic potentials in ischemia-reperfusion.

By combining a Hill-type pH probe and a pH-insensitive naphthalimide fluorophore, we synthesized a FRET-based ratiometric pH probe (PHHF), exhibiting a reduced pH transition width, representing a unique approach for development of sensitive probes for detection of biorelevant pH changes.

The balance between drug efficiency and its side effects on normal tissues is still a challenging problem to be solved in current cancer therapies. Among different strategies, cancer therapeutic methods based on nanomedicine delivery systems have received extensive attention due to their unique advantages such as improved circulation and reduced toxicity of drugs in the body. Herein, we constructed dual-responsive polymeric micelles DOXALS@MFM based on an upper critical solution temperature (UCST) polymer to simultaneously combine chemotherapy, photothermal therapy (PTT), and photodynamic therapy (PDT). Amphiphilic block copolymer P(AAm

Ischemia-reperfusion (I/R) injuries are from the secondary radicals of ONOO

Rationale: Nanoscale vehicles responsive to abnormal variation in tumor environment are promising for use in targeted delivery of therapeutic drugs specifically to tumor sites. Herein, we report the design and fabrication of self-accelerating H2O2-responsive plasmonic gold nanovesicles (GVs) encapsulated with tirapazamine (TPZ) and glucose oxidase (GOx) for synergistic chemo/starving therapy of cancers. Methods: Gold nanoparticles were modified with H2O2-responsive amphiphilic block copolymer PEG45-b-PABE330 by ligand exchange. The TPZ and GOx loaded GVs (TG-GVs) were prepared through the self-assembly of PEG45-b-PABE330 -grafted nanoparticles together with TPZ and GOx by solvent displacement method. Results: In response to H2O2 in tumor, the TG-GVs dissociate to release the payloads that are, otherwise, retained inside the vesicles for days without noticeable leakage. The released GOx enzymes catalyze the oxidation of glucose by oxygen in the tumor tissue to enhance the degree of hypoxia that subsequently triggers the reduction of hypoxia-activated pro-drug TPZ into highly toxic free radicals. The H2O2 generated in the GOx-catalyzed reaction also accelerate the dissociation of vesicles and hence the release rate of the cargoes in tumors. The drug-loaded GVs exhibit superior tumor inhibition efficacy in 4T1 tumor-bearing mice owing to the synergistic effect of chemo/starvation therapy, in addition to their use as contrast agents for computed tomography imaging of tumors. Conclusion: This nanoplatform may find application in managing tumors deeply trapped in viscera or other important tissues that are not compatible with external stimulus (e.g. light).

Malignant gliomas are the most common brain tumors, with generally dismal prognosis, early clinical deterioration and high mortality. Recently, 2-aminoquinoline scaffold derivatives have shown pronounced activity in central nervous system disorders. We herein reported a series of 2-aminoquinoline-3-carboxamides as novel non-alkylator anti-glioblastoma agents. The synthesized compounds showed comparable activity to cisplatin against glioblastoma cell line U87 MG in vitro. Among them, we found that 6a displayed good inhibitory activity against A172 and U118 MG glioblastoma cell lines and induced cell cycle arrest in the G2/M phase and apoptosis in U87 MG by flow cytometry analysis. Additionally, 6a displayed low cytotoxicity to several normal human cell lines. In silico study showed 6a had promising physicochemical properties and was predicted to cross the blood-brain barrier. Moreover, preliminary structure-activity relationships are also investigated, shedding light on further modifications towards more potent agents on this series of compounds. Our results suggest this compound has a promising potential as an anti-glioblastoma agent with a differential effect between tumor and non-malignant cells.

One hallmark of solid tumors, regardless of its type or stage, is the existence of an unsual acidic microenvironment, which has been considered a specific and ideal target for cancer imaging. Therefore, we developed a pH-activatable nanoprobe GNPs-CKL-FA for near-infrared fluorescence (NIR) and computed tomography (CT) imaging of tumors. This nanoprobe consists of a near-infrared fluorophore (Cy5.5), a pH-sensitive ketal linker, and gold nanoparticles (GNPs) decorated with folates that could bind to tumor cells' surface receptors to promote cellular internalization. This ability of folate to mediate tumor targeting and accelerate internalization has been confirmed by in vitro experiments with HeLa cells. The fluorescence of the nanoprobes successfully activated by low intracellular pH, especially in more acidic organelles. Furthermore, fluorescence signals increased to a greater extent when the pH in tumors was lowered by injection of acetate buffer and isoproterenol. The CT contrast of GNPs-CKL-FA was obtained after administering intravenously to HeLa subcutaneous tumor-bearing mice. These results suggest that GNPs-CKL-FA has the potential to be a pH-activatable fluorescent nanoprobe combined with CT contrast agent for tumor targeted imaging.

Antimicrobial resistance is a public health emergency and warrants coordinated global efforts. Challenge is that no alternative molecular platform has been identified for discovery of abundant antimicrobial hit compounds. Xanthene libraries have been screened for bioactive compounds. However, the potentially accessible chemistry space of xanthene dyes is limited by the existing xanthene synthesis. Herein we report a mild one-step synthesis, which permits late-stage introduction of a xanthene moiety onto i.e. natural products, pharmaceuticals, and bioactive compounds and construction of a focused library of rhodamine dyes exhibiting facile functional, topographical and stereochemical diversity. In vitro screening yields 37 analogs with mid-to-high bactericidal activity against WHO priority drug-resistant pathogens. These findings suggest that synthetic dye libraries exhibiting high structural diversity is a feasible chemical space combating antibacterial resistance, to complement the natural sources.

The fibroblast growth factor receptors (FGFRs) are increasingly considered attractive targets for therapeutic cancer intervention due to their roles in tumor metastasis and angiogenesis. Here, we identified a new selective FGFR inhibitor, C11, and assessed its antitumor activities. C11 was a selective FGFR1 inhibitor with an IC(50) of 19 nM among a panel of 20 tyrosine kinases. C11 inhibited cell proliferation in various tumors, particularly bladder cancer and breast cancer. C11 also inhibited breast cancer MDA-MB-231 cell migration and invasion via suppression of FGFR1 phosphorylation and its downstream signaling pathway. Suppression of matrix metalloproteinases 2/9 (MMP2/9) was associated with the anti-motility activity of C11. Furthermore, the anti-angiogenesis activity of C11 was verified in endothelial cells and chicken chorioallantoic membranes (CAMs). C11 inhibited the migration and tube formation of HMEC-1 endothelial cells and inhibited angiogenesis in a CAM assay. In sum, C11 is a novel selective FGFR1 inhibitor that exhibits potent activity against breast cancer metastasis and angiogenesis.

Photodynamic therapy (PDT) efficacy has been severely limited by the hypoxia in tumor microenvironment. A multitherapy modality was developed, integrating the advantages of each therapy and a nanocarrier: PDT and PDT-induced hypoxia-activated chemotherapy. Following PDT-induced hypoxia augmented in the periphery of the tumors, chemotherapy was locally activated. To this end, new indocyanine green (IR820) and a hypoxia-activated prodrug tirapazamine (TPZ) were loaded in glutathione (GSH) decomposable mesoporous organic silica nanoparticles (GMONs), tethered by hyaluronic acid (HA). This nanohybrid showed a tendency to accumulate and be retained in tumors, due to passive and active targeting. The IR820 produced singlet oxygen (1O2) under near-infrared (NIR) laser irradiation and concomitantly tumorous abnormality exacerbated hypoxia. TPZ-mediated hypoxia-activated chemotherapy acted to kill more tumor cells. In vivo results indicated that the tumor inhibition rate of dual-loaded nanohybrids was up to 76% under NIR laser irradiation. The immunofluorescence staining of tumor slices demonstrated that the superficial part of tumors experienced exacerbated hypoxia with laser irradiation, resulting in TPZ exerting powerful chemotherapy effects. This nanohybrid is expected to be valuable as spatiotemporally specific therapy for cancer.

Near-infrared dyes possess the qualities of lower interference with biological autofluorescence, low photon scattering, and deep tissue penetration, and are being increasingly involved in the development of biomaterials for sensing and precision medicine. However, dyes usually suffer from the disadvantages of poor water solubility and photobleaching, factors that limit their application in vivo. The introduction of supramolecular ensembles can provide an ideal solution. This review presents recently developed supramolecular ensembles modified by near-infrared dyes. Compared with small-molecule fluorophores, the specific size of a supramolecular-based fluorophore endows it with longer circulation time in the bloodstream, increasing its chances of reaching a specific target. In addition, the construction of supramolecule-based fluorophores with versatile functions can be achieved by simple encapsulation or doping, instead of by complicated chemical synthesis. Thus, supramolecular-complex-based fluorophores offer high potential in diagnosis and therapy. This review outlines four different species of near-infrared dye based ensembles in terms of their method of formation, including simple encapsulation or doping and copolymerisation. Recently, a new technology has employed modified fluorophores for in situ self-assembly that form supramolecular ensembles at a specific position, thus solving the problem of poor uptake of nanoparticles by cells, and is included in this review. Finally, the future of this field is considered.

Fluorescent probes find widespread applications in chemical analysis, biosensing, and disease diagnosis and are also the cornerstones of many cutting-edge biotechnologies. Yet, the numerous probes developed thus far are actually based on a handful of probe design principles. In this article, we wish to summarize the recent progress of the field toward the use of a novel "covalent-assembly" principle for probe design.

Glycoside hydrolase family 18 (GH18) chitinases play an important role in various organisms ranging from bacteria to mammals. Chitinase inhibitors have potential applications as pesticides, fungicides, and anti-asthmatics. Berberine, a plant-derived isoquinoline alkaloid, was previously reported to inhibit against various GH18 chitinases with only moderate K i values ranging between 20 and 70 μM. In this report, we present for the first time the berberine-complexed crystal structure of SmChiB, a model GH18 chitinase from the bacterium Serratia marcescens. Based on the berberine-binding mode, a hydrophobic cavity-based optimisation strategy was developed to increase their inhibitory activity. A series of berberine derivatives were designed and synthesised, and their inhibitory activities against GH18 chitinases were evaluated. The compound 4c showed 80-fold-elevated inhibitory activity against SmChiB and the human chitinase hAMCase with K i values at the sub-micromolar level. The mechanism of improved inhibitory activities was proposed. This work provides a new strategy for developing novel chitinase inhibitors.

Nitric oxide (NO) donors are valuable tools to probe the profound implications of NO in health and disease. The elusive nature of NO bio-relevance has largely limited the use of spontaneous NO donors and promoted the development of next generation NO donors, whose NO release is not only stimulated by a trigger, but also readily monitored via a judiciously built-in self-calibration mechanism. Light is without a doubt the most sensitive, versatile and biocompatible method of choice for both triggering and monitoring, for applications in complex biological matrices. Herein, we designed and synthesized an N-nitroso rhodamine derivative (NOD560) as a photo-triggered and photo-calibrated NO donor to address this need. NOD560 is essentially non-fluorescent. Upon irradiation by green light (532 nm), it efficiently release NO and a rhodamine dye, the dramatic fluorescence turn-on from which could be harnessed to conveniently monitor the localization, flux, and dose of NO release. The potentials of NOD560 for in vitro biological applications were also exemplified in in vitro biological models, i.e. mesenchymal stem cell (MSC) migration suppression. NOD560 is expected to complement the existing NO donors and find widespread applications in chemical biological studies.

Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.

The development of biocompatible two-photon fluorophores with a large absorption cross-section is challenging, despite the presence of theoretical guidelines. By rendering asymmetric PRODAN dye centrosymmetric, we designed and synthesized a novel class of two-photon fluorophores (TPZ). Their photophysical properties were investigated and their imaging potentials in cells, tissues and zebrafish were showcased.

Compared to normal cells, most cancer cells produce ATP by glycolysis under aerobic conditions rather than via the tricarboxylic acid cycle (TCA). This study is intended to determine whether 3B, a novel photosensitizer, can inhibit glycolysis and inflammation in breast cancer cells. We showed that 3B had the ability to repress glucose consumption as well as the generation of ATP, lactate and lactate dehydrogenase. 3B-PDT not only inhibited the expression of IL-1β and IL-6 but also affected the JAK-STAT3 inflammatory pathway in vitro. The present study showed that 3B featured a significant inhibitory effect on the expression of microRNA-155-5p and SOCS1 might serve as a target gene. In vivo studies revealed that 3B inhibited tumor growth and exhibited almost no side effects. Therefore, through the anti-glycolytic effect and breakage of the JAK/STAT3/SOCS1 feedback loop via miR-155-5p, 3B may potentially serve as a potential therapeutic agent against breast cancer.

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands.

Classic fluorescent dyes, such as coumarin, naphthalimide, fluorescein, BODIPY, rhodamine, and cyanines, are cornerstones of various spectroscopic and microscopic methods, which hold a prominent position in biological studies. We recently found that 9-amino-benzo[c]cinnoliniums make up a novel group of fluorophores that can be used in biological studies. They are featured with a succinct conjugative push-pull backbone, a broad absorption band, and a large Stokes shift. They are potentially useful as a small-molecule alternative to R-phycoerythrin to pair with fluorescein in multiplexing applications.

Summary The altered metabolism of tumor cells confers a selective advantage for survival and proliferation, and studies have shown that targeting such metabolic shifts may be a useful therapeutic strategy. We developed an intensely fluorescent, rapidly responsive, pH-resistant, genetically encoded sensor of wide dynamic range, denoted SoNar, for tracking cytosolic NAD + and NADH redox states in living cells and in vivo. SoNar responds to subtle perturbations of various pathways of energy metabolism in real time, and allowed high-throughput screening for new agents targeting tumor metabolism. Among > 5,500 unique compounds, we identified KP372-1 as a potent NQO1-mediated redox cycling agent that produced extreme oxidative stress, selectively induced cancer cell apoptosis, and effectively decreased tumor growth in vivo. This study demonstrates that genetically encoded sensor-based metabolic screening could serve as a valuable approach for drug discovery.

A novel polycyclic aromatic molecule, i.e. 1-oxo-1H-phenalene-2,3-dicarbonitrile (compound 1, initially misidentified as 8-oxo-8H-acenaphtho[1,2-b]pyrrolecarbonitrile) was discovered by our group in 2005. This parent compound is highlighted for its unique oxidative S(N)Ar(H) (nucleophilic substitution of aromatic hydrogen) reactivity that provides easy approaches to diverse derivatives with different long-wavelength fluorescence and important biological activities. To date, a large number of derivatives have been synthesized and evaluated by several international research groups, indicating the formation of a new and valuable family of functional chemicals. Some members have been functionalized for molecular or nanoparticle-based probes applicable in chemical and environmental sensing, biomolecule imaging and tumor diagnosis. Others have qualified as high potency anticancer agents specifically targeting different functional proteins in tumor cells. With regard to the increasing attention paid to this new chemical family, it is a good time to review major achievements in order to promote further and deeper investigation.

A novel fluorescent probe, E3, was designed, synthesized and evaluated. It responded to Co2+ with high selectivity and sensitivity under physiological neutral conditions specifically. Furthermore, notably, probe E3 was demonstrated to detect Co2+ in living cells, indicative of its practical application potential.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. The iNap sensors permitted quantification of cytosolic and mitochondrial NADPH pools that were controlled by cytosolic NAD+ kinase levels, and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We find that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase (G6PD) and AMP kinase (AMPK). Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live-cells, and gaining new insights into cell metabolism.

Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

In this study, a novel naphthalimide derivative 7c was designed which is topo II inhibiting though owning weak DNA binders. It was shown that 7c could induce cancer cells apoptosis and have less cytotoxicity in normal human cell. Further investigations on Hela cells revealed that 7c could also induce ROS generation, lysosome rupture as well as cathepsin B release. Subsequent mitochondrial damages including mitochondrial membrane permeabilization and the release of cytochrome c were also found in 7c when treating with Hela cells. According to our data, 7c may act as a lead compound for potential anticancer drugs. The idea of naphthalimides modification may also provide a novel strategy for naphthalimides design.

Endogenous vicinal-dithiol-containing proteins (VDPs) that have two thiol groups close to each other in space play a significant importance in maintaining the cellular redox microenvironment. Approaches to identify VDPs mainly rely on monitoring the different concentration of monothiol and total thiol groups or on indirect labeling of vicinal thiols by using p-aminophenylarsenoxide (PAO). Our previous work has reported the direct labeling of VDPs with a highly selective receptor PAO analogue, which could realize fluorescence detection of VDPs directly in living cells. Herein, we developed a conjugated approach to expand detectable tags to nitrobenzoxadiazole (NBD), fluorescein, naphthalimide, and biotin for the synthesis of a series of probes. Different linkers have also been introduced toward conjugation of VTA2 with these functional tags. These synthesized flexible probes with various features will offer new tools for the potential identification and visualization of vicinal dithiols existing in different regions of VDPs in living cells. These probes are convenient tools for proteomics studies of various disease-related VDPs and for the discovery of new drug targets.

A series of mono-, bis- and trisnaphthalimides with different substituents on the naphthalimide ring system was prepared. For compounds containing a thiophenyl substituent fluorescence spectroscopic measurements were performed and unexpectedly showed an increase in fluorescence emission for the bis- and trisnaphthalimide derivatives in phosphate buffered saline. Additional fluorescence microscopic experiments indicated an efficient cellular uptake of the thiophenyl substituted compound 1c into cultured tumor cells. Experiments on the inhibition of tumor cell proliferation were performed in MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cells and confirmed derivatives containing a nitro substituent as the most active compounds. Compound 1c demonstrated potential as photoinducable antitumor agent.

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P{sub 2} promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2.more » Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment. - Research Highlights: > B1 induced apoptosis in MCF-7 cells, following a transcriptional decrease in Bcl-2. > B1 treatment triggered p53 activation and leads to a p53-dependent down-regulation of Bcl-2. > B1 induced significant increase of p53 binding to Bcl-2 P{sub 2} promoter TATA box.« less

3-(dimethylamino-ethylamino)-8-oxo-8H-acenaphthol[1, 2-b]pyrrole-9-carboxylic acid (APCA), as a potent antitumor compound, showed anticancer activity on a series of established cancer cells. Meanwhile, the cytotoxic effects of APCA were much smaller on normal human cells than that on cancer cells. This study investigated the molecular mechanisms underlying APCA-induced growth inhibition in HeLa cells. The results showed that the APCA-induced cell cycle arrest at G(2)/M phase correlated with cyclinB1 and cyclin-dependent kinase 1 expression downregulation in a p53-independent manner, and also caused an increase in apoptosis, which was confirmed by characteristic morphological changes and increased apoptotic sub-G(1) population. Furthermore, translocation inhibition of nuclear factor-κB, upregulation of Bax, and downregulation of Bcl-2, caspase-3 and caspase-9 activation, and poly-(ADP-ribose) polymerase cleavage were observed in HeLa cells treated with APCA, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. In summary, APCA displayed an antitumor effect through cell cycle arrest and apoptotic induction in HeLa cells, which suggested that APCA might have therapeutic potential against cervix carcinoma as an effective lead compound.

We have connected a borondipyrromethene (BODIPY) donor to the 5' position of a tetramethylrhodamine (TMR) acceptor to form a high efficiency (over 99%) intramolecular fluorescence resonance energy transfer (FRET) cassette, BODIPY-rhodamine platform (BRP). While the good spectral overlap between the emission of BODIPY and the absorption of TMR was one favorable factor, another feature of this FRET system was the rigid and short biphenyl spacer that favored efficient through-bond energy transfer. More importantly, in this system, the 2'-carboxyl group of the rhodamine unit was preserved for the further modifications, which was as convenient as those carbonyl groups on the original rhodamines without connection to donors. For this reason, BRP is clearly differentiated from the previous ratiometric sensors based on donor rhodamine systems. To illustrate its value as a versatile platform, we introduced typical Hg(2+) receptors into BRP, through convenient one-pot reactions on the 2'-carboxyl group, and successfully developed two ratiometric sensors, BRP-1 and BRP-2, with different spirocyclic receptors that recognized Hg(2+) on different reaction mechanisms. Upon excitation at a single wavelength (488 nm), at which only BODIPY absorbed, both of the FRET sensors exhibited clear Hg(2+)-induced changes in the intensity ratio of the two strong emission bands of BODIPY and rhodamine. It should be noted that these ratiometric Hg(2+) sensors exhibited excellent sensitivity and selectivity Hg(2+), as well as pH insensitivity, which was similar to the corresponding 'turn-on' rhodamine sensors. While both ratiometric probes were applicable for Hg(2+) imaging in living cells, BRP-1 exhibited higher sensitivity and faster responses than BRP-2. Our investigation indicated that on a versatile platform, such as BRP, a large number of highly efficient ratiometric sensors for transition-metal ions could be conveniently developed.

Amonafide is a naphthalimide derivative with antitumor activity and has failed to enter clinical phase III, because of its high-variable and unpredictable toxicity. In order to develop selective, efficient, and safe drugs, applying the 'nonfused' aromatic system strategy, a series of 5-non-amino aromatic substituted naphthalimides as replacement for amonafide were designed and were synthesized from naphthalic anhydride by three steps including bromination, amination, and Pd(PPh₃)₄ catalyzed Suzuki reaction. These new naphthalimide derivatives, except 4b, not only exhibited better activity than amonafide against HeLa and P388D1 cell lines in vitro under the same experimental conditions, but also could avoid the side effect of amonafide due to their structure, which lacks an easy acetylated arylamine at the 5 position. The DNA-binding behavior of the naphthalimide derivatives was also investigated, and the results suggested that they bind to DNA via intercalation and 4a and 4g intercalated into DNA in different fashion.

Several 2-(substituted benzo[c,d]indol-2(1H)-ylidene)malononitriles have been designed and synthesized. Their DNA binding, antitumor and DNA damaging properties were evaluated. All the compounds exhibited efficient antitumor activities with preference to be against the tumor cell line 7721 rather than the tumor cell line MCF-7. Compound 1f could intercalate into DNA entirely presumably by the good conjugation of carbonyl group with benzo[c,d]indol moiety. What’s more, 1f exhibited potent toxicity against MCF-7 cells with IC50 at 0.003 μM and against 7721 cells at 0.115 μM, respectively.

Based on the advantages of multitarget drugs for cancer treatment, a new class of naphthalimides was designed, synthesized, and proved to inhibit topoisomerase II (topo II), induced lysosomal membrane permeabilization (LMP), and ultimately caused apoptosis and cell death. The majority of compounds 7a-d and 8a-d potently inhibited the growth of the five tested cancer cell lines with IC(50) values ranging from 2 to 10 microM and are more active than amonafide, a naphthalimide that was in phase III clinical trials. These compounds were tested for their interactions with DNA and their cell-free topo II inhibition activities, which demonstrated these compounds were weak DNA binders but modest topo II inhibitors. Furthermore, compounds 7b-d were found to notably induce LMP and exhibited better antiproliferative activity compared with their single-target analogues. All of the newly synthesized compounds were demonstrated to efficiently induce apoptosis via a mitochondrial pathway. Accordingly, a new paradigm was suggested for the design of novel multitarget anticancer drugs.

A family of 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid derivatives were synthesized as a result of our efforts to modify a series of acenaphthopyrrole aromatic-heterocycle compounds that proved to be potent anticancer drugs. Among the derivatives, 3d (3-(dimethylamino-propylamino)-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) and 3g (3-piperidine-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) showed potential anticancer activity and different action mechanism from our previously reported compounds. UV-vis absorption, circular dichroism and viscosity measurement indicated that effect of both compounds on the advanced DNA conformation was different, although they could bind to DNA in the same way. Cell cycle analysis showed that 3d could induce S-phase arrest followed by apoptosis, while 3g induced apoptosis. The results seem to imply that different action mechanism could contribute to the dissimilitude of biological activities toward 3d and 3g.

Naphthalimides, particularly amonafide and 2-(2-dimethylamino)-6-thia-2-aza-benzo[ def ]chrysene-1,3-diones (R16), have been identified to possess anticancer activities and to induce G 2 -M arrest through inhibiting topoisomerase II accompanied by Chk1 degradation. The current study was designed to precisely dissect the signaling pathway(s) responsible for the naphthalimide-induced cell cycle arrest in human colon carcinoma HCT116 cells. Using phosphorylated histone H3 and mitotic protein monoclonal 2 as mitosis markers, we first specified the G 2 arrest elicited by the R16 and amonafide. Then, R16 and amonafide were revealed to induce phosphorylation of the DNA damage sensor ataxia telangiectasia-mutated (ATM) responding to DNA double-strand breaks (DSBs). Inhibition of ATM by both the pharmacological inhibitor caffeine and the specific small interference RNA (siRNA) rescued the G 2 arrest elicited by R16, indicating its ATM-dependent characteristic. Furthermore, depletion of Chk2, but not Chk1 with their corresponding siRNA, statistically significantly reversed the R16- and amonafide-triggered G 2 arrest. Moreover, the naphthalimides phosphorylated Chk2 in an ATM-dependent manner but induced Chk1 degradation. These data indicate that R16 and amonafide preferentially used Chk2 as evidenced by the differential ATM-executed phosphorylation of Chk1 and Chk2. Thus, a clear signaling pathway can be established, in which ATM relays the DNA DSBs signaling triggered by the naphthalimides to the checkpoint kinases, predominantly to Chk2,which finally elicits G 2 arrest. The mechanistic elucidation not only favors the development of the naphthalimides as anticancer agents but also provides an alternative strategy of Chk2 inhibition to potentiate the anticancer activities of these agents.

A series of novel naphthalimide derivatives with flexible alkyl/aryl moieties were designed and synthesized. Their antitumor activities were evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8 and A375 cancer cell lines in vitro. The preliminary results showed that most of the derivatives had comparable antitumor activities over Amonafide with the IC(50) values of 10(-6) to 10(-5)M. More importantly, flow cytometric analysis indicated that the derivatives could effectively induce G(2)/M arrest and progress to apoptosis in HL-60 cell line after double staining with annexin V-FITC and propidium iodide. The present work provided a novel class of naphthalimide-based derivatives with potent apoptosis-inducing and antitumor activities for further optimization.

A new series of nitrogen-containing heterocycles 4 H -1,3,4-oxadiazin-5(6 H )-ones derivatives with hydrophobic and long chains were designed and synthesized by direct cyclization reaction of N ′-alkylation substituted aroylhydrazines with chloroacetyl chloride. The preliminary assays showed that some of the compounds displayed moderate to good inhibitory activities toward monoamine oxidase (MAO) at the concentration of 10 −5 –10 −3 M, and antitumor activities against human lung cancer A-549 and human prostate cancer PC-3 cell lines at μM level, which might provide new scaffold for anticancer agents. Furthermore, compounds 5i and 5m exhibited significant inhibitory activity on chitin biosynthesis, which might represent a novel class of highly potential inhibitors of chitin synthesis.

A series of novel naphthalimide derivatives with flexible leucine side chains were designed and synthesized. Their antitumor activities were evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8 and A375 cancer cell lines in vitro. The preliminary results showed that most of the derivatives had moderate antitumor activities with the IC 50 values of 10 −6 –10 −5 M. More importantly, compounds 8a – c exhibited exclusive antitumor activities against MCF-7 cell line. The interaction between compound 8b and BSA was also investigated. DNA binding experiments showed that these derivatives behaved as DNA intercalating agents. This work provided a novel class of naphthalimide-based lead compounds with exclusive antitumor activities against MCF-7 cell line for further optimization.

Several novel heterocyclic-fused naphthalimides intercalators with chiral amino side chains were investigated. Their side chains' chiral configuration determines DNA binding activities in the order: S-enantiomers > R-enantiomers. And their DNA photodamaging activities were in good agreement with their DNA binding constants, the S-enantiomers could photocleave circular supercoiled pBR322 DNA more efficiently than their R-enantiomers. S-enantiomer B(3) could photodamage DNA at 0.2 microM and cleave supercoiled plasmid DNA from form I to form II completely at 50 microM. Almost all of these intercalators showed effective cytoxicities against human lung cancer cells and murine leukemia cells. S-enantiomers showed different antitumor cytotoxicity by comparison with R-enantiomers. This work may provide additional information for the role of amino side chains on intercalators as antitumor agents.

A series of sulfur-substituted naphthalimides (1-5) was prepared and investigated as antitumor drugs. Initial DNA interaction studies (by the fluorescence quenching method, UV/vis and CD spectroscopy, thermal denaturation, topoisomerase Western blot analysis, and DNA photocleavage experiments) expectedly suggested the DNA and topoisomerase as main targets of the agents. Fluorescence spectroscopic and microscopic experiments indicated a significant sensitivity of the emission intensities of 3 and 5 to the cellular environment and confirmed the cellular uptake and biodistribution into cell compartments for 1-3 and 5. A comparative evaluation of the antiproliferative effects under different experimental setups (concerning drug exposure period and an additional short-time UV irradiation) revealed significant phototoxic effects for the environmentally sensitive compounds 3 and 5 and strongly suggested the further development of sulfur-substituted naphthalimides for potential use in photodynamic tumor therapy.

Novel tertiary amine N-oxides of naphthalimides were designed and synthesized as potential anticancer agents against hypoxic solid tumors. Although their ctDNA-binding affinities and cytotoxic activities against usual tumor cell lines were lower than those of corresponding amines, the N-oxides A1 and A4 showed hypoxia preference activities against A375 cells in vitro and might be used as interesting candidates of prodrug leads in hypoxic tumor cells.

A series of mono-1 H -benzo[ c , d ]indol-2-one with different amine side chains and bis-1 H -benzo[ c , d ]indol-2-one as novel family of DNA intercalators were designed and synthesized, the contributions of aromatic chromophores and amine side chains for DNA binding properties, for example, intercalation and electrostatic binding, respectively, were evaluated. Among them, A3 tailed with N , N -dimethylamino-ethyl-ethane-1,2-diamine showed selective anti-tumor activities against cell lines A549 and P388 with IC 50 0.428 μm and 1.69 μm.

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the present study, a novel amonafide analogue, 2-(2-dimethylamino)-6-thia-2-aza-benzo-[def]-chrysene-1,3-diones (R16) was synthesized by substituting 5′-NH2 of the naphthyl with a heterocyclic group to amonafide, with additional introduction of a thiol group. In a panel of various human tumor cell lines, R16 was more cytotoxic than its parent compound amonafide. It was also effective against multidrug-resistant cells. Importantly, the i.p. administration of R16 inhibited tumor growth in mice implanted with S-180 sarcoma and H22 hepatoma. The molecular and cellular machinery studies showed that the R16 functions as a topoisomerase II (topo II) poison via binding to the ATPase domain of human topo IIα. The superior cytotoxicity of R16 to amonafide was ascribed to its potent effects on trapping topo II–DNA cleavage complexes. Moreover, using a topo II catalytic inhibitor aclarubicin, ataxia-telangiectasia-mutated (ATM)/ATM- and Rad3-related (ATR) kinase inhibitor caffeine and topo II–deficient HL-60/MX2 cells, we further showed that R16-triggered DNA double-strand breaks, tumor cell cycle arrest, and apoptosis were in a topo II–dependent manner. Taken together, R16 stood out by its improved anticancer activity, appreciable anti–multidrug resistance activities, and well-defined topo II poisoning mechanisms, as comparable with the parent compound amonafide. All these collectively promise the potential value of R16 as an anticancer drug candidate, which deserves further development. [Mol Cancer Ther 2007;6(2):484–95]

A novel “turn-on” fluorescent probe HP for hypoxia imaging was designed and synthesized based on rhodamine B and a naphthalimide fluorophore. The fluorescence of HP is very weak owing to the FRET effect from rhodamine B to the azo-naphthalimide unit. Under hypoxia conditions, the azo-bond is reduced and the fluorescence at 581 nm enhances dramatically as a result of disintegration of the quencher structure. Verified by the cyclic voltammetry reduction potential and proposed product HPN, the probe HP could undergo the chemical and cytochrome P450 enzymatic reduction quickly. When cultured with HeLa cells, HP showed remarkable fluorescence differences at various oxygen concentrations, and the ratio of fluorescence intensity between hypoxic and normoxic cells could reach 9 fold.