Development And Applications Of NIR Fluorescent Probe For The Detection Of Carboxylesterase 1 Targeting Endoplasmic Reticulum

Objective:Developing an endoplasmic reticulum targeting,highly selective near-infrared fluorescent probe ERBM for the detection of CES1 activity;Characterizing the metabolic properties of the probe by in vitro assay method,and illustrating the metabolic characteristics of the probe mediate by CES1;Imaging the endogenous CES1 in living systems in vitro and in vivo;To reveal the changes of activity and expression of CES1under endoplasmic reticulum stress and acute liver injury;High-throughput screening of CES1 traditional Chinese medicine regulator by ERBM and to explore its efficacy;Therefore,ERBM is a promising fluorescent molecular tool to detect the function of CES1 in ER and CES1 metabolism related disease in clinic and provide useful guidance for clinical rational drug use.Methods:(1)Probe synthesis:According to the substrate preference of CES1,developed an ER targeted near-infrared fluorescent probe,ERBM,based on Bodipy fluorescent skeleton and endoplasmic reticulum targeted group p-toluene sulfonamide.(2)Illustrating the characteristics of metabolic progress of ERBM mediated by CES1,including enzyme linear,selectivity,chemical stability,and kinetic study.(3)Detecting the endogenous CES1 in living cells by fluorescence imaging,and confirming the ER targeting efficiency by colocalization imaging with commercial endoplasmic reticulum Tracker.(4)Imaging function of ERBM and Westren Blot were used to visually detect the changing pattern of CES1 under DTT-induced ER stress and APAP-induced acute liver injury.(5)ERBM was injected into mice for fluorescence imaging and established an acute liver injury model to explore the application of ERBM in in-vivo imaging and disease detection.(6)A high-throughput screening method was constructed to screen the traditional Chinese medicine regulators of CES1,and clopidogrel,the classical substrate of CES1,was used to evaluate whether there was a potential DDI between them.Results:Obvious fluorescence response was showed in 690 nm after the metabolism of ERBM by CES1,and good linear response of fluorescence intensity with enzyme concentration;ERBM showed highly selective toward CES1 after the hydrolases screening and chemical inhibition,it also exhibited good stability in a variety of metal ions and amino acids;the metabolic progress of ERBM mediated by CES1 followed a substrate-inhibition kinetic model and K_m=11.22±3.641μM;ERBM has good cell permeability,could real-time image CES1 in living cells;the imaging of CES1 using ERBM showed good co-localization with commercial endoplasmic reticulum Tracker;and the fluorescence signal of CES1 in LO2cells decreased significantly under ER stress,which were consistent with the expression of CES1 protein detected by WB;ERBM has good tissue permeability,could real-time image CES1 in mice;detection the changes of CES1 in acute liver injury model by ERBM,the results showed the characteristics of the down regulation of CES1,which were consistent with the expression of CES1 protein detected by WB.Using ERBM for high throughput screening,Rubia cordifolia L.showed excellently inhibitory activity,which could significantly inhibit the metabolic process of clopidogrel and Lycopus lucidus.,Phillyrin showed moderate inhibitory activity.Besides,pharmacokinetics of rats showed that clopidogrel combined with Rubia cordifolia L.could slow down the metabolic clearance process of clopidogrel and significantly increase the absorption rate and degree of clopidogrel.Conclusion:In summary,we have developed a near-infrared fluorescent probe ERBM which could target endoplasmic reticulum CES1.The assay results demonstrated that ERBM possessed the highly selective and sensitive toward CES1 and was successfully used for real-time imaing the activity of CES1 in living cells and animals,respectively.In addition,ERBM could tartet to the ER location and imgae the CES1 which was consistent with the commerial ER Tracker.At last,the characteristics of the down regulation of CES1under ER stress in LO2 cells and drug-induced acute liver injury model was revealed by ERBM imaging applicablity.Therefore,ERBM is expected to be a novel fluorescent molecular tool for exploring the biological function of CES1 and various related diseases.Simultaneously,the traditional Chinese medicine inhibitor-Rubia cordifolia L.combine with clopidogrel was used in clinical may generate drug-drug interaction,which can provide guidance basis for clinical rationalization of drug use.