Real-time identification of gut microbiota with aminopeptidase N using an activable NIR fluorescent probe

A NIR fluorescent probe(DDAA) derived from fluorophore DDAO with alanine as the recognition group was developed for sensing aminopeptidase N(APN) in gut microbiota.Using DDAA as the real-time guidance tool for the fluorescence imaging of intestinal microorganism,target bacteria and saccharomycete possessing active APN were identified successfully from human feces.

Sensing carboxylesterase 1 in living systems by a practical and isoformspecific fluorescent probe

Carboxylesterase 1(CES1), one of the most abundant serine hydrolases in mammals, has drawn much attentions in recent years, owing to this enzyme involves in many physiological processes via hydrolysis of both endogenous esters and xenobiotic esters. Herein, to real-time monitor the activities of CES1 in various biological systems, a practical and iso form-specific fluorescent probe was developed on the basis of the substrate preference of CES1, as well as the structural and optical properties of BODIPY dyes. After screening of a panel of BODIPY ester derivatives, probe 1 displayed the best combination of specificity,sensitivity, enzymatic kinetics and applicability for monitoring CES1 activities in real samples. This probe was successfully used to detect CESl activities in several biological systems including tissue preparations,living cells, tissue slices and zebrafish. Furthermore, the biomedical applications of probe 1 for screening of CES1 inhibitors were also demonstrated using tissue preparations or living cells as enzyme sources. In summary, a practical and broadly applicable tool for real-time monitoring CES1 in biological systems was developed and well-characterized, which held great promise for further investigations on CES1-associated drug discovery, clinical practice and fundamental research.

A highly selective fluorescent probe for real-time imaging of UDP-glucuronosyltransferase 1A8 in living cells and tissues

Uridine diphosphate(UDP)-glucuronosyltransferases(UGTs)are enzymes involved in the biotransformation of important endogenous compounds such as steroids,bile acids,and hormones as well as exogenous substances including drugs,environmental toxicants,and carcinogens.Here,a novel fluorescent probe BDMP was developed based on boron-dipyrromethene(BODIPY)with high sensitivity for the detection of UGT1A8.The glucuronidation of BDMP not only exhibited a redemission wavelength(lex/lem=500/580 nm),but also displayed an excellent UGT1A8-dependent fluorescence signal with a good linear relationship with UGT1A8 concentration.Based on this perfect biocompatibility and cell permeability,BDMP was successfully used to image endogenous UGT1A8 in human cancer cell lines(LoVo and HCT15)in real time.In addition,BDMP could also be used to visualize UGT1A8 in tumor tissues.These results suggested that BDMP is a promising molecular tool for the investigation of UGT1A8-mediated physiological function in humans.

A NIR fluorescent probe for Vanin-1 and its applications in imaging, kidney injury diagnosis,and the development of inhibitor

Vanin-1 is an amidohydrolase that catalyses the conversion of pantetheine into the aminothiol cysteamine and pantothenic acid(coenzyme A precursor), which plays a vital role in multiple physiological and pathological processes. In this study, an enzyme-activated near-infrared(NIR) fluorescent probe(DDAV) has been constructed for sensitively detecting Vanin-1 activity in complicated biosamples on the basis of its catalytic characteristics. DDAV exhibited a high selectivity and sensitivity toward Vanin-1 and was successfully applied to the early diagnosis of kidney injury in cisplatin-induced kidney injury model. In addition, DDAV could serve as a visual tool for in situ imaging endogenous Vanin-1 in vivo. More importantly, Enterococcus faecalis 20247 which possessed high expression of Vanin-1 was screened out from intestinal bacteria using DDAV, provided useful guidance for the rational use of NSAIDs in clinic. Finally, oleuropein as a potent natural inhibitor for Vanin-1 was discovered fromherbal medicines library using a high-throughput screening method using DDAV, which held great promise for clinical therapy of inflammatory bowel disease.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes

Currently,the development of selective fluorescent probes toward targeted enzymes is still a great challenge,due to the existence of numerous isoenzymes that share similar catalytic capacity.Herein,a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s)for cytochrome P450(CYP)which are key enzymes involving in metabolism of endogenous substances and drugs.In the first-stage of our filtering approach,near-infrared(NIR)fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system.In the second stage of our filtering approach,these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s).Using our double-filtering approach,probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2,respectively,to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis.These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.