Jingnan Cui

Addressing the lack of dynamic monitoring methods for assessing the combined toxicity of mixed pesticides, this study developed a fluorescent probe, CCHL, specifically responsive to leucine aminopeptidase (LAP). The probe utilized Cy7-COOH (CCH) as the fluorophore, with fluorescence recovery triggered by enzymatic hydrolysis. Spectral characterization confirmed a linear response between the probe and LAP activity within a concentration range of 0–0.9 μg/mL (R² = 0.992), along with excellent selectivity in the presence of coexisting biomolecules. Application experiments demonstrated that the combination of chlorfenapyr and beta-cyfluthrin significantly reduced LAP activity, revealing a notable antagonistic effect. The novel sensing strategy developed here provides a real-time, visualized analytical tool for evaluating the combined effects of mixed pollutants, demonstrating significant potential for environmental toxicology monitoring.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is an exopeptidase widely expressed on various cell surfaces that selectively cleaves N-terminal dipeptides from diverse substrates. In recent years, DPPIV inhibitors have been extensively utilized in the treatment of hepatitis mellitus (DM). In this study, we designed a far-red fluorescent probe, DBX-AP, through molecular docking simulations and by leveraging the functional characteristics of DPPIV. This probe enables rapid, highly selective, and real-time monitoring of DPPIV activity both in vitro and in vivo. Using DBX-AP, we developed a visual high-throughput screening technique for the detection of DPPIV inhibitors. From a library of 4828 compounds, three inhibitors (K784-2660, 6484-0066, and E699-0153) were identified for their strong inhibitory effects on DPPIV. These inhibitors not only suppressed DPPIV activity in the ileum of mice, thereby reducing GLP-1 degradation, but also effectively inhibited DPPIV activity in gut microbiota. The successful application of DBX-AP in visual detection technology highlights its potential for evaluating DPPIV activity and identifying novel DPPIV inhibitors for diabetes mellitus treatment.

Monoamine oxidase A (MAO-A) is a dimeric flavoprotein that is found in the mitochondrial membrane. Currently, there is a lack of near-infrared fluorescent probes (NIR-FPs) with good specificity and high sensitivity for detecting MAO-A, making it difficult to accurately recognize and image cells in vitro and in vivo. In this study, the NIR-FP DDM-NH₂ was designed and synthesized in order to detect MAO-A specifically in live biological systems. The probe comprised two functional components: dicyanoisophosphone as an NIR dye precursor and alanine as a recognition moiety. After identifying MAO-A, the probe exhibited an NIR emission peak at 770 nm with a significant Stokes shift (180 nm), 11-fold response factor, low detection limit of 99.7 nM, and considerably higher affinity toward MAO-A than that toward MAO-B, indicating high sensitivity. In addition, DDM-NH₂ was effective when applied to the image-based assessment of MAO-A activity in HeLa cells, zebrafish, and tumor-bearing mice, demonstrating great potential for visualization-based research and MAO-A application in vivo.

Tuberculosis (TB) is a chronic systemic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Methionine aminopeptidase 1 (MtMET-AP1) is a hydrolase that mediates the necessary post-translational N-terminal methionine excision (NME) of peptides during protein synthesis, which is necessary for bacterial proliferation and is a potential target for the treatment of tuberculosis. Based on the functional characteristics of MtMET-AP1, we developed an enzymatic activated near-infrared fluorescent probe DDAN-MT for rapid, highly selective, and real-time monitoring of endogenous MtMET-AP1 activity in M. tuberculosis. Using the probe DDAN-MT, a visually high-throughput screening technique was established, which obtained three potential inhibitors (GSK-J4 hydrochchloride, JX06, and lavendustin C) against MtMET-AP1 from a 2560 compounds library. More importantly, these inhibitors could inhibit the growth of M. tuberculosis H37Ra especially (MICs < 5 mu M), with low toxicities on intestinal bacteria strains and human cells. Therefore, the visual sensing of MtMET-AP1 was successfully performed by DDAN-MT, and MtMET-AP1 inhibitors were discovered as potential antituberculosis agents.

A novel near-infrared (NIR) fluorescent probe CHC-CES1 based on a hemi-cyanine skeleton for detecting carboxylesterase 1 (CES1) activity was developed. Herein, CHC-CES1 could be specifically hydrolysed to CHC-COOH along with a significant NIR fluorescence signal enhancement at 670 nm. Systematic evaluation indicated that CHC-CES1 possessed an outstanding selectivity and sensitivity towards CES1, and possessed good chemical stability in complex biosamples. Finally, CHC-CES1 was successfully used for the real-time imaging of endogenous CES1 activity in living cells. Moreover, CHC-CES1 was applied to evaluate the inhibitory effects of various pesticides towards CES1, and visually revealed the inhibitory effect of combined residue pesticides.

An endoplasmic reticulum targeting NIR fluorescent probe (ERBM) was developed for real-time monitoring of carboxylesterase 1 (CES1) and exhibited excellent ER location in living cell imaging. In addition, ERBM was applied to illustrate the regulation characteristics of CES1 under ER stress and acute liver injury models at the cell and animal level.

Lipase found in the gut microbiota participates in the digestion and absorption of dietary fats. As such, the gut microbiota is involved in the regulation of the host metabolism, affecting the levels of lipids and free fatty acids, ultimately resulting in obesity. In this study, an enzymatic activatable near-infrared fluorescent probe, DDAO-C6, was developed for visually sensing endogenous lipase from gut microbes. Using DDAO-C6, a cultivated intestinal yeast strain was rapidly identified from human feces that exhibited high lipase expression and was identified as Trichosporon asahii Y2. We then determined that the colonization of the gut of mice with T. asahii Y2 increased lipase activity in the digestive tract and promoted obesity and hyperlipidemia when the mice were fed high fat diets. Above all, the present research resulted in a fluorescence visualization tool for the functional investigation of gut microbiota associated with obesity and disorders of lipid metabolism.

CYP2J2 as an endoplasmic reticulum (ER)-expressed vital cytochrome P450 isoform participates in the metabolism of endogenous polyunsaturated fatty acids. Its abnormal expression and function are closely related to the progress of cancer and cardiovascular diseases. Herein, an ER-targeting near-infrared (NIR) fluorescent probe ER-BnXPI was developed for monitoring CYP2J2 activity, which possessed a high selectivity and sensitivity toward CYP2J2 among various CYP450 isoforms and exhibited excellent subcellular localization for ER. Then, the CYP2J2 variation behavior under the ER stress model was imaged by ER-BnXPI in living cells and successfully used for the in vivo imaging in different tumors that well distinguished tumor tissues from para-cancerous tissues. All these findings fully demonstrated that ER-BnXPI could be used as a promising tool for exploring the physiological function of CYP2J2 and provided some novel approach for the diagnosis and therapy of CYP2J2-related vascular inflammation and cancer.