A dual-targeting fluorescent probe for simultaneous and discriminative visualization of lipid droplets and endoplasmic reticulum.

A single fluorescent probe (SF-probe) that can simultaneously and discriminatively visualize two organelles is a powerful tool to investigate their interaction in cellular processes. However, it is still challenging to develop this unique type of fluorescent probe due to the lack of a feasible design strategy. Herein, we proposed a dual-targeting group strategy to construct SF-probes by integrating two different organelle-targeting groups into the same fluorophore. A versatile fluorophore and two nonintrusive organelle-targeting groups are elements of this strategy. In view of only a few SF-probes having been developed for the simultaneous and discriminative imaging of lipid droplets (LDs) and endoplasmic reticulum (ER), as a proof of concept, a SF-probe, LDER, was designed and synthesized by introducing an LD targeting group and an ER targeting group onto the 1,8-naphthalimide fluorophore. Owing to the specific structure of the fluorophore, both targeting groups at two terminals of 1,8-naphthalimide can fully play their respective roles without mutual interference. Furthermore, the ability of the two groups to target their respective targets is comparable, enabling LDER to bind LDs and ER evenly. Meanwhile, LDER is very susceptible to polarity, which is advantageous for the discriminative imaging of LDs and ER. In addition, the interaction between LDs and ER was investigated.

Indomethacin-induced spectral responses of naphthalimide-based dyes to serum albumin: effects of substituent and spacer.

Four indomethacin-naphthalimide binaries with different proton receptors at 4-position of naphthalimide were designed and synthesized. N,N-Dimethylethylenediamine and N-methyl piperazine were served as proton receptors as well as solubility regulators. Indomethacin, an inhibitor for cyclooxygenase-2 overexpressed on cancer cells, was connected at the imine N through different spacers. The attachment of indomethacin significantly quenched the fluorescence of all compounds with obvious red-shift in the absorption maxima due to the strong photo-induced electron transfer process of the folded-state. Human serum albumin (HSA) triggered about 15-fold fluorescence enhancements of DMN-IMC-5 with 30 nm blue-shift. However, it caused much smaller fluorescence increments of other compounds, suggesting that indomethacin, the linker and proton receptor play critical roles in HSA identification. Fluorescence bioimaging results show that indomethacin enables the naphthalimide-based compounds to fluorescent imaging living cells. Molecular docking reveals that the introduction of indomethacin improved the binding affinity of the dyes to HSA.

Photodiagnosis and photodynamic effects of bacteriochlorin-naphthalimide conjugates on tumor cells and mouse model.

Photo-induced cytotoxicity and antitumor activity of a series of dual function agents for photodynamic therapy (PDT) and fluorescent imaging based on bacteriochlorin photosensitizer conjugated with various naphthalimide fluorophores was studied in vitro using murine tumor cells of S37 sarcoma and in vivo on mice bearing murine S37 sarcoma. Upon irradiation at the absorption maximum of the photosensitizer, the activity of conjugates was as high as in the case of individual bacteriochlorin, while an additional excitation of the naphthalimide fragment led to an increase in the PDT efficacy due to resonance energy transfer from the fluorophore to photosensitizer. The fluorescence contrast and specific cytotoxic activity measurements indicate that the conjugate of bacteriochlorin with 3,4-dimethoxestyrene-substituted naphthalimide is the most promising agent for the application as theranostic in PDT.

A Golgi Apparatus-Targeting, Naphthalimide-Based Fluorescent Molecular Probe for the Selective Sensing of Formaldehyde.

Formaldehyde (FA) is a colorless, flammable, foul-smelling chemical used in building materials and in the production of numerous household chemical goods. Herein, a fluorescent chemosensor for FA is designed and prepared using a selective organ-targeting probe containing naphthalimide as a fluorophore and hydrazine as a FA-binding site. The amine group of the hydrazine reacts with FA to form a double bond and this condensation reaction is accompanied by a shift in the absorption band of the probe from 438 nm to 443 nm upon the addition of FA. Further, the addition of FA is shown to enhance the emission band at 532 nm relative to the very weak fluorescent emission of the probe itself. Moreover, a high specificity is demonstrated towards FA over other competing analytes such as the calcium ion (Ca2+), magnesium ion (Mg2+), acetaldehyde, benzaldehyde, salicylaldehyde, glucose, glutathione, sodium sulfide (Na2S), sodium hydrosulfide (NaHS), hydrogen peroxide (H2O2), and the tert-butylhydroperoxide radical. A typical two-photon dye incorporated into the probe provides intense fluorescence upon excitation at 800 nm, thus demonstrating potential application as a two-photon fluorescent probe for FA sensing. Furthermore, the probe is shown to exhibit a fast response time for the sensing of FA at room temperature and to facilitate intense fluorescence imaging of breast cancer cells upon exposure to FA, thus demonstrating its potential application for the monitoring of FA in living cells. Moreover, the presence of the phenylsulfonamide group allows the probe to visualize dynamic changes in the targeted Golgi apparatus. Hence, the as-designed probe is expected to open up new possibilities for unique interactions with organ-specific biological molecules with potential application in early cancer cell diagnosis.

Disentangling the Structure-Activity Relationships of Naphthalene Diimides as Anticancer G-Quadruplex-Targeting Drugs.

In the context of developing efficient anticancer therapies aimed at eradicating any sort of tumors, G-quadruplexes represent excellent targets. Small molecules able to interact with G-quadruplexes can interfere with cell pathways specific of tumors and common to all cancers. Naphthalene diimides (NDIs) are among the most promising, putative anticancer G-quadruplex-targeting drugs, due to their ability to simultaneously target multiple G-quadruplexes and their strong, selective in vitro and in vivo anticancer activity. Here, all the available biophysical, biological, and structural data concerning NDIs targeting G-quadruplexes were systematically analyzed. Structure-activity correlations were obtained by analyzing biophysical data of their interactions with G-quadruplex targets and control duplex structures, in parallel to biological data concerning the antiproliferative activity of NDIs on cancer and normal cells. In addition, NDI binding modes to G-quadruplexes were discussed in consideration of the structures and properties of NDIs by in-depth analysis of the available structural models of G-quadruplex/NDI complexes.

A mitochondria-targeted fluorescent dye naphthalimide-thioether-cyanine for NIR-activated photodynamic treatment of cancer cells.

In this work, an NIR-activated fluorescent dye naphthalimide-thioether-cyanine (NPSCY) was developed for the photodynamic treatment of cancer cells. In this dye, naphthalimide and cyanine were selected as the two fluorophores, which were linked by the thioether group. Under 660 nm irradiation, NPSCY could produce 1O2 rapidly, suggesting the potential for photodynamic therapy. Cys can be considered as one of the markers of cancer cells and NPSCY could distinguish Cys from three channels (433 nm, 475 nm, 733 nm) due to the bilateral recognition of the thioether group, which was helpful for accurately locating cancer cells. Fortunately, NPSCY could also produce 1O2 after being reacted with the intracellular biological thiols, which also avoided the inactivation of the photosensitizer in cancer cells. The co-localization coefficient of 0.873 indicated that the cyanine group promoted the aggregation of NPSCY in mitochondria. This photosensitizer showed low dark toxicity and high phototoxicity. Meanwhile, the half-maximal inhibitory concentration (IC50) was calculated to be 3.7 µM. NPSCY could inhibit cell migration after irradiation at 660 nm.

Simultaneous Zn2+ tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells.

Zn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.

Electric-Field-Mediated Electron Tunneling of Supramolecular Naphthalimide Nanostructures for Biomimetic H2 Production.

The design and synthesis of two semiconducting bis (4-ethynyl-bridging 1, 8-naphthalimide) bolaamphiphiles (BENI-COO- and BENI-NH3+ ) to fabricate supramolecular metal-insulator-semiconductor (MIS) nanostructures for biomimetic hydrogen evolution under visible light irradiation is presented. A H2 evolution rate of ca. 3.12 mmol g-1 ⋅h-1 and an apparent quantum efficiency (AQE) of ca. 1.63 % at 400 nm were achieved over the BENI-COO- -NH3+ -Ni MIS photosystem prepared by electrostatic self-assembly of BENI-COO- with the opposite-charged DuBois-Ni catalysts. The hot electrons of photoexcited BENI-COO- nanofibers were tunneled to the molecular Ni collectors across a salt bridge and an alkyl region of 2.2-2.5 nm length at a rate of 6.10×108  s-1 , which is five times larger than the BENI-NH3+ nanoribbons (1.17×108  s-1 ). The electric field benefited significantly the electron tunneling dynamics and compensated the charge-separated states insufficient in the BENI-COO- nanofibers.

Enzyme Instructed Self-assembly of Naphthalimide-dipeptide: Spontaneous Transformation from Nanosphere to Nanotubular Structures that Induces Hydrogelation.

Understanding the structure-morphology relationships of self-assembled nanostructures is crucial for developing materials with the desired chemical and biological functions. Here, phosphate-based naphthalimide (NI) derivatives have been developed for the first time to study the enzyme-instructed self-assembly process. Self-assembly of simple amino acid derivative NI-Yp resulted in non-specific amorphous aggregates in the presence of alkaline phosphatase enzyme. On the other hand, NI-FYp dipeptide forms spherical nanoparticles under aqueous conditions which slowly transformed into partially unzipped nanotubular structures during the enzymatic catalytic process through multiple stages which subsequently resulted in hydrogelation. The self-assembly is driven by the formation of ß-sheet type structures stabilized by offset aromatic stacking of NI core and hydrogen bonding interactions which is confirmed with PXRD, Congo-red staining and molecular mechanical calculations. We propose a mechanism for the self-assembly process based on TEM and spectroscopic data. The nanotubular structures of NI-FYp precursor exhibited higher cytotoxicity to human breast cancer cells and human cervical cancer cells when compared to the nanofiber structures of the similar Fmoc-derivative. Overall this study provides a new understanding of the supramolecular self-assembly of small-molecular-weight hydrogelators.

Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKß) with a similar potency (Ki = 0.35 µM for CaMKKα, and Ki = 0.2 µM for CaMKKß) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 µM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

Synthesis of naphthalimide derivatives with potential anticancer activity, their comparative ds- and G-quadruplex-DNA binding studies and related biological activities.

Two new cytotoxic 1,8-naphthalimide derivatives have been synthesized and characterized. Their biological activities as cytotoxicity and antimicrobial activities and inhibitory activities against DNA-polymerase were evaluated. The interactions of compounds with double-stranded- and quadruple-DNA have been studied by UV-Vis, fluorescent intercalator displacement, competition dialysis, circular dichroism and the findings were compared with the parent naphthalimide and the other compounds. The results show that both compounds (1 and 2) and the parent compound NI have strong cytotoxic activities against Beas-2B, MCF-7, HepG2 and MDA-MB-231 cancer cell lines, antimicrobial activities against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and inhibitory activities towards Taq-polymerase and transcriptase. These novel cationic compounds 1 and 2 can stabilize G-quadruplexes DNA according to thermal denaturation experiments, they change the 3D structure of the DNA (see details in CD experiments) and they exhibit different binding affinities for q-DNA and ds-DNA revealed by spectrophotometric titrations and competitive dialysis studies.

Achieving the ratiometric imaging of steroid sulfatase in living cells and tissues with a two-photon fluorescent probe.

Herein, a novel two-photon ratiometric fluorescence assay was proposed for monitoring endogenous steroid sulfatase (STS) activity, which could be applied for the ratiometric imaging of STS activity in the endoplasmic reticulum of living cells and tissues and also could be used to distinguish estrogen-dependent tumor cells from other types of cells.

Cancer-Specific hNQO1-Responsive Biocompatible Naphthalimides Providing a Rapid Fluorescent Turn-On with an Enhanced Enzyme Affinity.

Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is overexpressed in cancer cells and associated with the drug resistance factor of cancer. The objective of this work is the development of fluorescent probes for the efficient detection of hNQO1 activity in cancer cells, which can be employed for the cancer diagnosis and therapeutic agent development. Herein, we report naphthalimide-based fluorescent probes 1 and 2 that can detect hNQO1. For hNQO1 activity, the probes showed a significant fluorescence increase at 540 nm. In addition, probe 1, the naphthalimide containing a triphenylphosphonium salt, showed an enhanced enzyme efficiency and rapid detection under a physiological condition. The detection ability of probe 1 was superior to that of other previously reported probes. Moreover, probe 1 was less cytotoxic during the cancer cell imaging and readily provided a strong fluorescence in hNQO1-overexpressed cancer cells (A549). We proposed that probe 1 can be used to detect hNQO1 expression in live cells and it will be applied to develop the diagnosis and customized treatment of hNQO1-related disease.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

Directing Traffic: Halogen-Bond-Mediated Membrane Transport.

The plasma membrane regulates the transport of molecules into the cell. Small hydrophobic molecules can diffuse directly across the lipid bilayer. However, larger molecules require specific transporters for their entry into the cell. Regulating the cellular entry of small molecules and proteins is a challenging task. The introduction of halogen, particularly iodine, to small molecules and proteins is emerging to be a promising strategy to improve the cellular uptake. Recent studies reveal that a simple substitution of hydrogen atom with iodine not only increases the cellular uptake, but also regulates the membrane transport. The strong halogen-bond-forming ability of iodine atoms plays a crucial role in the transport and the introduction of iodine may provide an efficient strategy for studying membrane activity and cellular functions and improving the delivery of therapeutic agents. This Concept article does not provide a comprehensive picture of membrane transport but highlights halogen-substitution as a novel strategy for understanding and regulating the cell-membrane traffic.

Insight into the delivery channel and selectivity of multiple binding sites in bovine serum albumin towards naphthalimide-polyamine derivatives.

Naphthalimide derivatives are types of small-molecule anticancer drug candidates; however, their negative factors and potential side effects make their application limited. The pharmacophores select a direct access into the tumor cells as the first choice; this can reduce the side effect of the anti-cancer drugs on the normal cells. Herein, the delivery and binding of the naphthalimide-polyamine complex assisted by the bovine serum albumin (BSA) protein have been studied by combining several molecular dynamic simulations. The plausible transportation channels and the most favorable pathways for the delivery of the naphthalimide-polyamine complex to two drug sites (DSI and DSII), their thermodynamic and dynamic properties and the mechanisms have been discussed in detail. The residues His287 and Phe394 acted as guards in the DSI and DSII, respectively, which played a gating-switch role by flipping the ring from open to close during the compound delivery. The binding mode, binding energy and substituent effects have been also identified. The two drug sites have different preferences towards the compound with the electron-withdrawing and electron-donating substituents, and their strong interactions are more sensitive to the number of the substituent groups. The naphthalimide-polyamine complexes are more likely to choose DSI, both thermodynamically and dynamically, as compared to DSII. This selective specificity of these two drug sites manipulated by the electron-withdrawing and electron-donating substituents is quite promising for the design of new naphthalimide drugs.

1,8-Naphthalimide-Substituted BODIPY Dyads: Synthesis, Structure, Properties, and Live-Cell Imaging.

A set of 1,8-naphthalimide (NPI)-substituted 4,4-difluoroboradiaza-s-indacene (BODIPY) dyads 1 a-1 c were designed and synthesized by the Pd-catalyzed Sonogashira cross-coupling reaction of ethynyl substituted NPI 1 with the meso-, ß-, and α-halogenated BODIPYs a, b, and c, respectively. The BODIPY 1 c exhibits redshifted absorption, which suggests better electronic communication with substitution at the α-position of BODIPY compared with at the meso and ß positions, which was further supported by time-dependent DFT calculations. The optical band gap follows the order 1 a>1 b>1 c. The single-crystal X-ray structures of dyads 1 a-1 c are reported, which reflect planar orientations of the BODIPY units with respect to the NPIs. The DFT-optimized structures show good correlation with the experimental data obtained from the single-crystal X-ray structures. The packing diagram of 1 a shows a sheet-like arrangement, 1 b forms a ladder-like structural motif, and 1 c forms a complex 3D structural arrangement. The dyads 1 a-1 c show low cytotoxicity (IC50 >100 µm). The confocal microscopy studies with HeLa and A375 cells (when treated with dyads 1 a-1 c) show that all the dyads easily enter the cell membrane and show significant multicolor intracellular fluorescence covering the entire visible range with clear emissions in blue, green, and red channels.

A Novel Mechanism of Inactivating Antibacterial Nitro Compounds in the Human Pathogen Staphylococcus aureus by Overexpression of a NADH-Dependent Flavin Nitroreductase.

Recently, the nitro-substituted bisquaternary bisnaphthalimides were reported to have substantial anti-infective activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Here, we selected resistant S. aureus clones by cultivation in increasing concentrations of the most active compound, MT02. Interestingly, MT02-resistant variants induced a diffusible red color of the broth. Chromatographic and spectroscopic investigations revealed a stepwise reduction of the bisquaternary bisnaphthalimides' nitro groups to amino groups. The corresponding derivatives were completely inactive against staphylococci. RNA sequencing experiments revealed a strong overexpression of a novel oxidoreductase in MT02-resistant strains. Deletion mutants of this enzyme did not produce the red color and were not able to develop resistance against bisquaternary bisnaphthalimides. Biochemical reactions confirmed an NADH-dependent deactivation of the nitro-substituted compounds. Thus, this is the first report of a nitroreductase-based antibiotic resistance mechanism in the human pathogen S. aureus.

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach.

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.

Glycosidase activated release of fluorescent 1,8-naphthalimide probes for tumor cell imaging from glycosylated 'pro-probes'.

Glycosylated 4-amino-1,8-naphthalimide derivatives possess a native glycosidic linkage that can be selectively hydrolysed in situ by glycosidase enzymes to release the naphthalimide as a fluorescent imaging or therapeutic agent. In vitro studies using a variety of cancer cell lines demonstrated that the naphthalimides only get taken up into cells upon enzymatic cleavage from the glycan unit; a mechanism that offers a novel approach for the targeted delivery of probes/drugs.