Simultaneous detection of mitochondrial viscosity and peroxynitrite in livers from subjects with drug-induced fatty liver disease using a novel fluorescent probe.

Drug-induced fatty liver disease (DIFLD) is a basic clinicopathological example of drug-induced liver injury (DILI). Some drugs can inhibit ß-oxidation in hepatocyte mitochondria, leading to steatosis in the liver. Additionally, drug-induced inhibition of ß-oxidation and the electron transport chain (ETC) can lead to increased production of reactive oxygen species (ROS) such as peroxynitrite (ONOO-). Therefore, it is reasonable to suspect that compared to a healthy liver, viscosity and ONOO- levels are elevated in livers during DIFLD. A novel, smart, dual-response fluorescent probe-Mito-VO-was designed and synthesized for the simultaneous detection of viscosity and ONOO- content. This probe had a large emission shift of 293 nm and was capable of monitoring the viscosity of, and the ONOO- content in, cell and animal models alike, either individually or simultaneously. For the first time, Mito-VO was successfully used to demonstrate the elevated viscosity and the amount of ONOO- in livers from mice with DIFLD.

A Water-soluble Near-infrared Fluorescent Probe for Cysteine/Homocysteine and Its Application in Live Cells and Mice.

A near-infrared (NIR) and water-soluble probe was synthesized and studied for the detection of Cys/Hcy in aqueous solution, living cells and mice. The probe was composed of cyanine derivative as the NIR fluorescent reporting unit and pyrimidiny-thioether moiety as the Cys/Hcy responsive unit. Treatment with Cys/Hcy induced the formation of sulfur-substituted products, then intramolecular rearrangement reaction would occur to produce amino-substituted products and resulting in enhanced red fluorescence emissions. It could be applied to sense Cys/Hcy both in solution with the detection limit of 0.17 µM (or 0.32 µM) and in living cells. Cell imaging experiments proved that such a probe exhibited good cell penetration. In addition, the probe could detect Cys/Hcy in live mice with strong turn-on fluorescent response.

Novel cascade reaction-based fluorescent cyanine chemosensor for cysteine detection and bioimaging in living system.

Precise detection of intracellular Cys will be helpful to accurately assess the physiological functions in the physiological and pathological processes. Herein, a new probe Meoeth-Cy-OBz-oCl capable of Cys sensing with high selectivity over other mercaptoamino-acid molecules including Hcy and GSH was developed. The studies on sensing mechanism supported thiols-induced SNAr substitution-rearrangement cascade reaction which allowed discriminating Cys from Hcy/GSH. And its preferential fluorescence response of Meoeth-Cy-OBz-oCl to intracellular Cys was also achieved by means of fluorescence imaging in HeLa cells. Besides, Meoeth-Cy-OBz-oCl was confirmed possessing mitochondria-targeting ability in living cells. In addition, fluorescence imaging in BALB/c mice revealed that Meoeth-Cy-OBz-oCl could visually monitor the variation of Cys in vivo.

A Reaction-Based Fluorescent Probe for Imaging of Native Hypochlorous Acid.

Hypochlorous acid (HOCl), one of the reactive oxygen species (ROS), is highly reactive and short-lived. It is a challenge to dynamic monitor HOCl activity in living systems. Hence, we synthesized a new fluoresce nt probe RF1 based on protection of the hydroxyl group by N,N-dimethylthiocarbamate recognition group, which reached a low fluorescence background signal and highly sensitive property. On account of the electrophilic addition of Cl+ to the sulfide of thiocarbamate moiety, probe RF1 was converted to resorufin and triggered emitting bright. RF1 showed not only the highly sensitive and selective response to HOCl in vitro, but also can be applied in environmental water samples and detected HOCl by test strips. Besides, the ability of RF1 monitoring HOCl in HeLa cells by exogenous simulation and tracing native HOCl in macrophages cells were also explored.

Fluorescence detection of intracellular pH changes in the mitochondria-associated process of mitophagy using a hemicyanine-based fluorescent probe.

Intracellular pH behaves as a vital parameter in the physiological and pathological processes. Novel small molecule probes for precise and dynamic monitoring of pH fluctuations in cellular physiological processes are still highly required. Herein, we present a hemicyanine-based probe (HcPH) detection of the pH changes during the intracellular process of mitochondria-associated autophagy. HcP-H exhibits highly reversible and ratiometric fluorescence detection of pH variation due to the deprotonation/protonation process, showing orange fluorescence (λem = 557 nm) in basic media (pH 8.0) and green fluorescence (λem = 530 nm) in acidic media (pH 6.2), respectively. Organelle localization experiment in HeLa cells demonstrates that this probe could selectively accumulate in mitochondria, showing almost overlap with that of Mito-Tracker Green FM. More importantly, Fluorescence imaging of HcP-H in HeLa cells subjected to the nutrient deprivation has demonstrated that this probe could monitor the intracellular pH changes in the mitochondria-associated process of mitophagy. It is clearly confirmed that HcP-H would serve as a promising fluorescent probe for tracing mitophagy in living cells.

Synthesis and biological evaluation of quinoxaline derivatives as tubulin polymerization inhibitors that elevate intracellular ROS and triggers apoptosis via mitochondrial pathway.

A series of novel quinoxaline derivatives were synthesized and evaluated for their antiproliferative activity in three human cancer cell lines. Compound 12 exhibited the most potent antiproliferative activity with IC50 in the range of 0.19-0.51 µM. The compound inhibited tubulin polymerization and disrupted the microtubule network, leading to G2/M phase arrest. Furthermore, compound 12 induced ROS production and malfunction of mitochondrial membrane potential. Compound 12 led to cancer cells apoptosis in a dose-dependent manner. Western blot analysis showed that compound 12 induced up-regulation of p21 and affected the expression of cell cycle-related proteins. The binding mode was also probed by molecular docking.

A novel NBD-based fluorescent turn-on probe for the detection of cysteine and homocysteine in living cells.

Biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are involved in a number of biological processes and play crucial roles in biological systems. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, we developed a new turn-on fluorescent probe 1 based on 7-nitro-2,1,3-benzoxadiazole (NBD) with high selectivity and sensitivity for Cys/Hcy on account of nucleophilic substitution and Smiles rearrangement reaction. The probe could sense Cys/Hcy rapidly, the intensity of fluorescence increased immediately within 1min. Furthermore, the probe is low toxic and has been successfully applied to detect intracellular Cys/Hcy by cell fluorescence imaging in living normal and cancer cells.

A dual-selective fluorescent probe for discriminating glutathione and homocysteine simultaneously.

Homocysteine (Hcy) and glutathione (GSH) play important roles in a variety of physiological and pathological processes. Abnormal levels of Hcy and GSH are related to various diseases. Fluorescent probes for detecting them with sensitive and selective are highly desirable. However, efficient discrimination of Hcy and GSH is still a challenge for their similar molecular structures and chemical properties. Herein, we report a naphthalimide and sulfonyl benzoxadiazole (SBD) based dual-selective fluorescent probe for Hcy and GSH over other amino acids. The probe exhibited weak fluorescence (Φ = 0.075, in DMSO) at 490 nm and fluorescence enhancement upon addition of GSH (1-20 µM) with a detection limit of 0.8 µM. The probe also exhibited ratiometric fluorescence responses for Hcy (fluorescence at 490 nm decreased and fluorescence at 552 nm increased). The fluorescence intensity ratio (I552/I490) showed a good linear correlation with the Hcy concentrations in range of 3-20 µM and the detection limit was 0.1 µM. Moreover, this probe was successfully utilized for monitoring Hcy and GSH in living cells.

Synthesis and biological evaluation of N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxylic acid derivatives as tubulin polymerization inhibitors.

A series of novel N-substituted 3-oxo-1,2,3,4-tetrahydro-quinoxaline-6-carboxy- lic acid derivatives were synthesized and evaluated for their biological activities. Among all synthesized target compounds, 13d exhibited the most potent antiproliferative activity against HeLa, SMMC-7721, K562 cell line (IC50 = 0.126 µM, 0.071 µM, 0.164 µM, respectively). Furthermore, compound 13d inhibited tubulin polymerization (IC50 = 3.97 µM), arrested cell cycle at the G2/M phase and induced apoptosis. The binding mode at the colchicine binding site was also probed. These studies provided a new molecular scaffold for the further development of antitumor agents that target tubulin.

A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity.

Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.