The Development Of Fluorescent Probe For Carboxylesterase 1

Serine hydrolases,which is named due to its catalytic site containing serine.As a member of serine hydrolases superfamily,carboxylesterase?CES,EC:3.1.1.1?plays important roles in metabolic elimination of xenobiotic drugs and toxicant,and also promote the hydrolysis of endogenic lipids.As the two major CES in mammalian,both of CES1 and CES2 participate in the hydrolysis of esters?amides and thioesters,but display different substrates preference and discrepant distribution.CES1 expresses in liver and prefer to hydrolyze substrate with large acyl group and small alcohol group such as clopidogrel,while CES2 is majorly express in intestine and more likely to metabolize compound with small acyl group and large alcohol group such as capecitabine.There are many methods including western blotting,rt-PCR and proteomics have been used to detect enzyme in human body,but these methods need complex operations and expensive equipment.In contrast,fluorescent probe have been widely used in monitoring enzyme activity due to highly selective?sensitive and easy management.At present,available fluorescent probe for CES are majorly belong to CES2,only BMBT(?_em=480 nm)is a selective probe for CES1.In this paper,4 BODIPY fluorescent probe were synthesized on the basis of substrate preference of CES1.After preliminary screening,probe 1 displayed the best combination of selectivity and sensitivity toward CES1,and chemical inhibition assays,reaction phenotype assays further validated the selectivity of probe 1 toward CES1.Enzyme kinetic assays suggested that CES1 has good affinity toward probe 1?Km<5?M?,which was the major enzyme involved in the metabolism of CES1 in HLMs.Furthermore,we also established the standard curve of fluorescent intensities increased with CES1 concentrations and incubation time,the fluorescent intensities at 560 nm displayed a good linear correlation?R²>0.98?with CES1 concentrations and incubation time when CES1 concentration and incubation time up to2?g/mL and 30 min,respectively.Based on above findings,we used probe 1 and clopidogrel?a known substrate for CES1?to detect CES1 activities in individual HLMs and plasma respectively,and correlation analysis of the hydrolytic rates probe 1 and clopidogrel displayed good correlation?R²>0.95?.then,with probe 1 as substrate,we achieved imaging CES1activities in living cells,tissues slice?the depth of imaging up to 40?m?and zebrafish.We also assessed the inhibition abilities of CES1 inhibitors?BNPP and UKA?in recombinant CES1,HLMs and living cells as enzyme sources.Both BNPP and UKA displayed good inhibition effects?IC50<200 nM?and similar inhibition abilities,but BNPP showed stronger inhibition than UKA in living cells,this results suggested that probe 1 could be used to screen and assess CES1 inhibitors in multiple levels.In a word,a highly selective and sensitive fluorescent probe for CES1 has been developed,which can promote research in CES1-related physiological and pathological process.