Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation.

In this study, two "on-off" probes (BF2-cur-Ben and BF2-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF2-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (Km = 16 ± 1.6 µM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF2-cur-Ben forms more hydrogen bonds (seven, while BF2-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of Km values. These two probes could enable recognition of intracellular AChE and probe BF2-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF2-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.

Design, synthesis, and evaluation of a carboxylesterase detection probe with therapeutic effects.

In this study, a lysosomal targeting fluorescent probe recognition on CEs was designed and synthesized. The obtained probe BF2-cur-Mor demonstrated excellent selectivity, sensitivity, pH-independence, and enzyme affinity towards CEs within 5 min. BF2-cur-Mor could enable recognition of intracellular CEs and elucidate that the CEs content of different cancer cells follows the rule of HepG2 > HCT-116 > A549 > HeLa, and the CEs expression level of hepatoma cancer cells far exceeds that of normal hepatic cells, being in good agreement with the previous reports. The ability of BF2-cur-Mor to monitor CEs in vivo was confirmed by zebrafish experiment. BF2-cur-Mor exhibits some pharmacological activity in that it can induce apoptosis in hepatocellular carcinoma cells but is weaker in normal hepatocyte cells, being expected to be a potential "diagnostic and therapeutic integration" tool for the clinical diagnosis of CEs-related diseases.

Design, synthesis and biological evaluation of novel dual-targeting fluorescent probes for detection of Fe3+ in the lysosomes of hepatocytes mediated by galactose-morpholine moieties.

In this work, novel dual-targeting probes composed of galactose and morpholine were designed and synthesized for monitoring Fe3+ levels in the lysosome of hepatocyte. MP-Gal-1, MP-Gal-2 and MP-Gal-3 showed good selectivity and sensitivities toward Fe3+ with the detection limits of 9.40 × 10-8 M, 7.68 × 10-8 M and 7.10 × 10-8 M, respectively. 1:2 stoichiometry is the most likely recognition mode between probe and Fe3+. Low toxic MP-Gal-1, MP-Gal-2 and MP-Gal-3 exhibited favorable hepatic targeting effect in both cell and tissue levels, which was because the galactose group of probe could be recognized by ASGPR overexpressed on the hepatocytes. The hepatocyte-targeting capacity followed MP-Gal-1 < MP-Gal-2 < MP-Gal-3 trend, which was attributed to the galactose cluster effect. MP-Gal-1, MP-Gal-2 and MP-Gal-3 also displayed good lysosomes-targeting capacities, because the basic morpholine moiety of probes could be easily attracted by the acidic lysosome. Therefore, MP-Gal-1, MP-Gal-2 and MP-Gal-3 have good dual targeting capacities (liver and lysosome) and could be used to detect lysosomal Fe3+ in the liver, which is great significant for precise diagnosis and treatment of liver lysosomal iron-related diseases.

Anethole Attenuates Enterotoxigenic Escherichia coli-Induced Intestinal Barrier Disruption and Intestinal Inflammation via Modification of TLR Signaling and Intestinal Microbiota.

This study aimed to investigate the effects of dietary anethole supplementation on the growth performance, intestinal barrier function, inflammatory response, and intestinal microbiota of piglets challenged with enterotoxigenic Escherichia coli K88. Thirty-six weaned piglets (24 ± 1 days old) were randomly allocated into four treatment groups: (1) sham challenge (CON); (2) Escherichia coli K88 challenge (ETEC); (3) Escherichia coli K88 challenge + antibiotics (ATB); and (4) Escherichia coli K88 challenge + anethole (AN). On day 12, the piglets in the ETEC, ATB, and AN group were challenged with 10 mL E. coli K88 (5 × 109 CFU/mL), whereas the piglets in the CON group were orally injected with 10 mL nutrient broth. On day 19, all the piglets were euthanized for sample collection. The results showed that the feed conversion ratio (FCR) was increased in the Escherichia coli K88-challenged piglets, which was reversed by the administration of antibiotics or anethole (P < 0.05). The duodenum and jejunum of the piglets in ETEC group exhibited greater villous atrophy and intestinal morphology disruption than those of the piglets in CON, ATB, and AN groups (P < 0.05). Administration of anethole protected intestinal barrier function and upregulated mucosal layer (mRNA expression of mucin-1 in the jejunum) and tight junction proteins (protein abundance of ZO-1 and Claudin-1 in the ileum) of the piglets challenged with Escherichia coli K88 (P < 0.05). In addition, administration of antibiotics or anethole numerically reduced the plasma concentrations of IL-1ß and TNF-α (P < 0.1) and decreased the mRNA expression of TLR5, TLR9, MyD88, IL-1ß, TNF-α, IL-6, and IL-10 in the jejunum of the piglets after challenge with Escherichia coli K88 (P < 0.05). Dietary anethole supplementation enriched the abundance of beneficial flora in the intestines of the piglets. In summary, anethole can improve the growth performance of weaned piglets infected by ETEC through attenuating intestinal barrier disruption and intestinal inflammation.

Development of Asialoglycoprotein-Mediated Hepatocyte-Targeting Antitumor Prodrugs Triggered by Glutathione.

One antitumor ß-elemene derivative W-105 and three novel hepatocyte-targeting prodrugs (W-1-5, W-2-9, and W-3-8) were designed and synthesized. W-105 (IC50 6.107 µM) could cause cell apoptosis through upregulating the activity of caspase-3. The hepatocyte-targeting capacities of the aimed compounds followed the W-105 (parent compound) < W-1-5 (monodentate-galactose) < W-2-9 (bidentate-galactose) < W-3-8 (tridentate-galactose) order, which is attributed to the excellent affinity of the galactose ligand to ASGPR and the galactose-cluster recognition effect. Furthermore, prodrugs W-3-8 exhibited good antitumor activity and low toxic side effects. The liquid chromatography-mass spectrometry (LC-MS) assays revealed that prodrugs (W-1-5, W-2-9, and W-3-8) could release the antitumor pharmacophore in the presence of GSH (mimic the condition of the tumor cell) and maintain the low-toxic structures in the absence of GSH (mimic the condition of the normal cell). The release mechanisms of prodrugs were also proposed. Overall, these prodrugs developed in this study had potential in the treatment of liver cancer.