CN114478578B - Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof - Google Patents
Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof Download PDFInfo
- Publication number
- CN114478578B CN114478578B CN202111597847.9A CN202111597847A CN114478578B CN 114478578 B CN114478578 B CN 114478578B CN 202111597847 A CN202111597847 A CN 202111597847A CN 114478578 B CN114478578 B CN 114478578B
- Authority
- CN
- China
- Prior art keywords
- ces1
- probe substrate
- carboxylesterase
- reaction
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102100030817 Liver carboxylesterase 1 Human genes 0.000 title claims abstract description 148
- 239000000523 sample Substances 0.000 title claims abstract description 90
- 239000000758 substrate Substances 0.000 title claims abstract description 88
- 101710201076 Carboxylesterase 1 Proteins 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000005415 bioluminescence Methods 0.000 title claims description 16
- 230000029918 bioluminescence Effects 0.000 title claims description 16
- 230000000694 effects Effects 0.000 claims abstract description 51
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 9
- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 21
- 108060001084 Luciferase Proteins 0.000 claims description 15
- 239000005089 Luciferase Substances 0.000 claims description 15
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 14
- 239000003112 inhibitor Substances 0.000 claims description 14
- 238000006460 hydrolysis reaction Methods 0.000 claims description 13
- 230000007062 hydrolysis Effects 0.000 claims description 11
- 238000011534 incubation Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 229940126214 compound 3 Drugs 0.000 claims description 6
- 238000011156 evaluation Methods 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 229940125782 compound 2 Drugs 0.000 claims description 4
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- 238000005886 esterification reaction Methods 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 3
- 238000006467 substitution reaction Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000000411 inducer Substances 0.000 claims description 2
- 101000938676 Homo sapiens Liver carboxylesterase 1 Proteins 0.000 abstract description 126
- 238000001514 detection method Methods 0.000 abstract description 44
- 102000004190 Enzymes Human genes 0.000 abstract description 38
- 108090000790 Enzymes Proteins 0.000 abstract description 38
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 6
- 241000894007 species Species 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 230000002503 metabolic effect Effects 0.000 abstract description 4
- 230000004907 flux Effects 0.000 abstract description 3
- 238000010171 animal model Methods 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 69
- 229940088598 enzyme Drugs 0.000 description 37
- 210000004027 cell Anatomy 0.000 description 29
- 150000001875 compounds Chemical class 0.000 description 26
- 210000001519 tissue Anatomy 0.000 description 26
- 230000005764 inhibitory process Effects 0.000 description 20
- 239000000047 product Substances 0.000 description 19
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 10
- 238000003384 imaging method Methods 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000001035 drying Methods 0.000 description 9
- 239000011734 sodium Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 7
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 7
- 101100172720 Rattus norvegicus Ces1e gene Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 210000004185 liver Anatomy 0.000 description 7
- QGJZLNKBHJESQX-UHFFFAOYSA-N 3-Epi-Betulin-Saeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(=C)C)C5C4CCC3C21C QGJZLNKBHJESQX-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 101100170933 Arabidopsis thaliana DMT1 gene Proteins 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 125000002947 alkylene group Chemical group 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000004020 luminiscence type Methods 0.000 description 5
- 239000002207 metabolite Substances 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- CLOUCVRNYSHRCF-UHFFFAOYSA-N 3beta-Hydroxy-20(29)-Lupen-3,27-oic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C(O)=O)CCC5(C)CCC(C(=C)C)C5C4CCC3C21C CLOUCVRNYSHRCF-UHFFFAOYSA-N 0.000 description 4
- MIJYXULNPSFWEK-GTOFXWBISA-N 3beta-hydroxyolean-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CCC(C)(C)C[C@H]5C4=CC[C@@H]3[C@]21C MIJYXULNPSFWEK-GTOFXWBISA-N 0.000 description 4
- DIZWSDNSTNAYHK-XGWVBXMLSA-N Betulinic acid Natural products CC(=C)[C@@H]1C[C@H]([C@H]2CC[C@]3(C)[C@H](CC[C@@H]4[C@@]5(C)CC[C@H](O)C(C)(C)[C@@H]5CC[C@@]34C)[C@@H]12)C(=O)O DIZWSDNSTNAYHK-XGWVBXMLSA-N 0.000 description 4
- XUJNEKJLAYXESH-UWTATZPHSA-N D-Cysteine Chemical compound SC[C@@H](N)C(O)=O XUJNEKJLAYXESH-UWTATZPHSA-N 0.000 description 4
- 229930195710 D‐cysteine Natural products 0.000 description 4
- JKLISIRFYWXLQG-UHFFFAOYSA-N Epioleonolsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4CCC3C21C JKLISIRFYWXLQG-UHFFFAOYSA-N 0.000 description 4
- 206010067125 Liver injury Diseases 0.000 description 4
- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 description 4
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 description 4
- QGJZLNKBHJESQX-FZFNOLFKSA-N betulinic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C QGJZLNKBHJESQX-FZFNOLFKSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- PZXJOHSZQAEJFE-UHFFFAOYSA-N dihydrobetulinic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(C)C)C5C4CCC3C21C PZXJOHSZQAEJFE-UHFFFAOYSA-N 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 231100000753 hepatic injury Toxicity 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 210000001589 microsome Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- MQYXUWHLBZFQQO-UHFFFAOYSA-N nepehinol Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C(=C)C)C5C4CCC3C21C MQYXUWHLBZFQQO-UHFFFAOYSA-N 0.000 description 4
- 229940100243 oleanolic acid Drugs 0.000 description 4
- HZLWUYJLOIAQFC-UHFFFAOYSA-N prosapogenin PS-A Natural products C12CC(C)(C)CCC2(C(O)=O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OCC(O)C(O)C1O HZLWUYJLOIAQFC-UHFFFAOYSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000011158 quantitative evaluation Methods 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- JBDOSUUXMYMWQH-UHFFFAOYSA-N 1-naphthyl isothiocyanate Chemical compound C1=CC=C2C(N=C=S)=CC=CC2=C1 JBDOSUUXMYMWQH-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 3
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 3
- 108010053652 Butyrylcholinesterase Proteins 0.000 description 3
- 102100032404 Cholinesterase Human genes 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- JGHIPJWMIOZUBZ-UHFFFAOYSA-N [2-(1,3-benzothiazol-2-yl)-6-methoxyphenyl] benzoate Chemical compound COC1=CC=CC(C=2SC3=CC=CC=C3N=2)=C1OC(=O)C1=CC=CC=C1 JGHIPJWMIOZUBZ-UHFFFAOYSA-N 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 3
- 229960003009 clopidogrel Drugs 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 102000054098 human CES1 Human genes 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- QGJZLNKBHJESQX-ULZDWRHHSA-N (1r,3as,5ar,5br,7ar,9r,11ar,11br,13ar,13br)-9-hydroxy-5a,5b,8,8,11a-pentamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysene-3a-carboxylic acid Chemical compound C1C[C@@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C QGJZLNKBHJESQX-ULZDWRHHSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 102000003846 Carbonic anhydrases Human genes 0.000 description 2
- 108090000209 Carbonic anhydrases Proteins 0.000 description 2
- 102000013392 Carboxylesterase Human genes 0.000 description 2
- 108010051152 Carboxylesterase Proteins 0.000 description 2
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 2
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 2
- 102000006378 Catechol O-methyltransferase Human genes 0.000 description 2
- 108020002739 Catechol O-methyltransferase Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101001134456 Homo sapiens Pancreatic triacylglycerol lipase Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 235000006550 Liquidambar Nutrition 0.000 description 2
- 241000208682 Liquidambar Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000010504 bond cleavage reaction Methods 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 102000046759 human PNLIP Human genes 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 210000001853 liver microsome Anatomy 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000011896 sensitive detection Methods 0.000 description 2
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 238000010025 steaming Methods 0.000 description 2
- -1 thioester compounds Chemical class 0.000 description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102100033639 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102100036806 Carboxylesterase 5A Human genes 0.000 description 1
- 102000010970 Connexin Human genes 0.000 description 1
- 108050001175 Connexin Proteins 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000016354 Glucuronosyltransferase Human genes 0.000 description 1
- 108010092364 Glucuronosyltransferase Proteins 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- 101000898006 Homo sapiens Cocaine esterase Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- MHSVUSZEHNVFKW-UHFFFAOYSA-N bis-4-nitrophenyl phosphate Chemical compound C=1C=C([N+]([O-])=O)C=CC=1OP(=O)(O)OC1=CC=C([N+]([O-])=O)C=C1 MHSVUSZEHNVFKW-UHFFFAOYSA-N 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 208000030224 brain astrocytoma Diseases 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 1
- 125000004452 carbocyclyl group Chemical group 0.000 description 1
- 239000002327 cardiovascular agent Substances 0.000 description 1
- 229940125692 cardiovascular agent Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 229940125898 compound 5 Drugs 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 150000002194 fatty esters Chemical class 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 102000055467 human CES2 Human genes 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 125000002249 indol-2-yl group Chemical group [H]C1=C([H])C([H])=C2N([H])C([*])=C([H])C2=C1[H] 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000004694 iodide salts Chemical group 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000010517 secondary reaction Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
- C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
- C07D513/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and discloses a specific fluorescent probe substrate for measuring carboxylesterase 1 activity, and a preparation method and application thereof. The probe substrate can be used for quantitative detection of CES1 enzyme activity in cell or tissue samples from different species, can also be used for rapid screening of CES1 modulators, and can be used as a probe substrate of CES1 of experimental animals in vivo and in whole to evaluate individual and species differences of metabolic enzyme CES1. The invention also discloses a preparation method of the probe substrate. The CES1 fluorescent probe substrate provided by the invention has the advantages of high specificity, high sensitivity, high detection convenience and high flux detection, has different detection modes compared with a fluorescent probe, is not interfered by biological matrixes basically, and has higher selectivity and accuracy in detecting CES1 single enzyme in vitro.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a specific bioluminescence probe substrate for measuring carboxylesterase 1, and a preparation method and application of the probe.
Background
Carboxylesterase 1 (carboxyleseterase 1,CES1,EC 3.1.1.1) belongs to the serine hydrolase family and is widely available in mammals. CES1 is predominantly distributed in the liver in humans and exhibits significant tissue specificity. CES1 is localized to the endoplasmic reticulum in subcellular organelles, with C-terminal connexins responsible for immobilization of CES1 to the endoplasmic reticulum, and its active center located at the N-terminal end of the protein. CES1 favors hydrolysis of ester, amide and thioester compounds of large acyl linked small alcohol group structures, which are key enzymes for ester-catalyzed metabolism. Most carboxylesterase substrate drugs are esters (e.g., lu Fei amide, irinotecan and capestatin), thioesters, amides and carbamates are potential substrates for carboxylesterases. Most cardiovascular drugs, statins and phenoxy acids are substrates for carboxylesterases. More than 80% of clopidogrel is metabolically inactivated by CES1, and the activity of CES1 directly influences the concentration of active products of clopidogrel in vivo and influences the effectiveness and safety of clopidogrel administration. CES1 is a key enzyme in endogenous metabolism for cholesterol ester and fatty ester metabolism. These are associated with disorders of lipid metabolism. CES1 was found to be closely related to the occurrence and development of various liver lipid metabolism diseases, CES1 knockout mice cause increased liver production and increased hepatic low density lipoprotein secretion, and cause hyperlipidemia and increased fat deposition in peripheral tissues. Detection of CES1 activity provides guidance for studying the metabolic processes of drugs in vivo.
Because of the specific and high distribution of CES1 in the liver, we hypothesize that when hepatic cells are damaged, CES1 in the cells may be released to the extracellular and even blood circulatory system as a marker such as transaminase, and the analysis of liver-derived molecules in the system is an important way to perform non-invasive tests. CES1 is exocrine to the cytosol or remains hydrolytically active, and because of this feature CES1 activity can be characterized doubly by residual activity in liver tissue and CES1 activity in blood or extracellular fluids, CES1 activity characterization methods can characterize the percentage of residual and hepatocyte destruction, above that illustrates CES1 is likely to be an evaluation index of liver injury.
The main detection methods of CES1 at present are immunoblotting, proteomics, ultraviolet spectroscopy, capillary electrophoresis and fluorescence detection. Immunoblotting is usually based on specific binding of antigen-antibody, and this method is usually excellent in specificity, but the antibody needs to be stored at low temperature, is easy to inactivate after multiple uses, and is expensive. Proteomics methods require specific instruments LC-MS and are quantified by detecting specific peptide fragments after proteolysis. This method has expensive instruments and complicated operation steps, and as with the immunization method, only the absolute content of the enzyme can be quantified or semi-quantified. It has been previously reported that the protein expression of CEs is not always positively correlated with its activity. The ultraviolet spectrum rule is based on the change of absorbance after hydrolysis to express the enzyme activity, and the specificity of the substrates is poor and can not be used for detecting the CES1 activity in a complex system. Still other substrates are detected by liquid phase detection. Compared with other technologies, the fluorescence detection method has the advantages of high sensitivity, small wound and real-time imaging, and has great advantages in analyzing CES1 in cells and tissues. The CES1 probe substrate with high sensitivity and strong specificity is developed, so that not only can the important role of CES1 in related diseases be better explored, but also powerful technical support can be provided for screening CES1 target drugs and quantitatively measuring CES1 activity in a biological system.
Currently, fluorescent probes used for measuring CES1 mainly comprise 2- (benzothiazol-2-yl) -6-methoxyphenyl benzoate (BMBT) and D-methyl fluorescein (DME) bioluminescence probes, wherein the probe substrate of the BMBT ratio can realize quantitative detection of CES1 in biological samples. However, this probe substrate has poor selectivity and is hydrolyzed by a protein having an ester bond hydrolyzing function such as albumin in addition to CES1. And the maximum emission wavelength of the product is 488nm, and the detection is easily interfered by biological matrixes. Separation by means of high performance liquid chromatography coupled fluorescence detectors is required to achieve CES1 functional analysis of complex samples. Bioluminescent probe DME has great advantage in quantifying CES1 in a variety of complex biological systems, such as cell and tissue preparations, but the probe has some drawbacks in selectivity. At equal protein concentrations, the selectivity of DME for CES1 and butyrylcholinesterase was about 30-fold, which means that in samples with a more abundant butyrylcholinesterase content (e.g. plasma/serum), the quantitative results of CES1 activity using DME were not reliable. In addition, hydroxyl groups in DME molecules are easily metabolized by the important drug metabolizing enzyme glucuronyl transferase (UGTs) in humans, and the presence of UGTs affects the detection results in living cell, living tissue or in vivo studies.
The above-identified drawback is that DME is utilized to further explore the limitations of CES1 function. Therefore, development of a CES1 probe reaction with high selectivity and a high-throughput detection method matched with the CES1 probe reaction have important practical values.
Disclosure of Invention
The invention aims to provide a specific bioluminescence probe substrate for measuring carboxylesterase 1 (CES 1) activity, which has a different detection mode compared with a fluorescent probe, is basically interfered with 0 by a biological matrix, and has higher selectivity and sensitivity compared with the prior bioluminescence probe, and an application thereof. The distribution and the function of CES1 in various biological systems can be quantitatively evaluated by utilizing the probe reaction.
The invention is realized by the following technical scheme:
a specific fluorogenic probe substrate for measuring carboxylesterase 1 (CES 1), which can be specifically catalyzed by CES1 to generate ester bond hydrolysis reaction and generate corresponding (S) -2- (6, 7-dihydro- [4,5-f ] indole-thiazolyl) -4, 5-dihydrothiazole-4-formic acid derivative (DDC for short), wherein the structural general formula of the probe is as follows:
wherein R1 is selected from H, alkyl, - (C) 1 -C 8 Alkylene) -carboxyl groups, - (C 1 -C 8 Alkylene) -ester groups, - (C 1 -C 8 Alkylene) -amino, - (C 1 -C 8 Alkylene) -cyano, - (C 1 -C 8 Alkylene) -nitro, - (C 1 -C 3 Alkylene) -O- (C 1 -C 3 Alkyl), carbocyclyl, - (C) 1 -C 3 Alkylene) -carbocyclyl,Aryl, - (C) 1 -C 3 Alkylene) -aryl, heteroaryl, - (C 1 -C 3 Alkylene) -heteroaryl, heterocyclyl or- (C 1 -C 3 Alkylene) -heterocyclyl. R is R 2 Selected from C 1 -C 10 An alkyl group.
Preferably, R is H or C1-C6 alkyl, more preferably H, methyl, ethyl, propyl or isopropyl.
The structure of the hydrolyzed product is as follows:
in a preferred embodiment of the present invention, when r1=h, r2=ch 3 When the probe substrate is:
(S) -2- (6, 7-dihydro- [4, 5-f)]Indole-thiazolyl) -4, 5-dihydrothiazole-4-carboxylic acid methyl ester (methyl (S) -2- (6, 7-dihydro-5H-thiazolo [4, 5-f)]Indol-2-yl) -4, 5-dihydrothiazole-4-carbonyl, abbreviated as DDM; or r1=ch 3， R2＝CH 3 (S) -2- (7-methyl-6, 7-dihydro- [4, 5-f)]Indole-thiazolyl) -4, 5-dihydrothiazole-4-carboxylic acid methyl ester (DDM-2).
The invention also provides a preparation method of the specific bioluminescence probe substrate for measuring carboxylesterase 1 (CES 1) activity, which comprises the following steps:
1) Performing cyano substitution reaction on the compound 2 to obtain a compound 3;
2) Cyano hydrolysis of compound 3 with D-cysteine and base to the corresponding acid (compound 4);
3) The esterification reaction is carried out to produce a specific fluorogenic probe substrate (compound 5) for measuring carboxylesterase 1.
In the step 1), the compound 2 and cyanide salt are added with a catalyst, reacted at 120-140 ℃ until the reaction is completed, and the compound 3 is obtained by extraction and washing; the molar ratio of the compound 2 to cyanide ions in cyanide salt is 1:1-3, preferably 1:2; the catalyst is an iodide salt, preferably NaI. The solvent used was DMSO.
In step 2), the molar ratio of compound 3 to D-cysteine is 1:1-3:1-2, preferably 1:2:1.5; occurs; the alkali is carbonate or bicarbonate; the solvent used is an alcohol, preferably methanol.
In the step 3), the compound 4 and methanol are subjected to esterification reaction, preferably EDCI and DMAP are used as catalysts; the solvent used was dichloromethane.
The specific fluorescent probe substrate can be used for detecting carboxylesterase 1, and qualitatively or quantitatively detecting the activity of the carboxylesterase 1.
A method for detecting carboxylesterase 1 comprises mixing the specific fluorescent probe substrate with a sample to be detected, performing enzymatic reaction, and detecting fluorescence intensity to qualitatively or quantitatively detect CES1 activity. The activity of CES1 in different biological systems can be quantitatively determined by quantitatively detecting the substrate elimination rate per unit time or the production rate of its carboxyl product.
The measuring method comprises the following steps:
adding a fluorogenic probe substrate for measuring the specificity of carboxylesterase 1 into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of an incubation system is 5.5-10.5, and adding luciferase to start bioluminescence reaction, wherein the reaction time is 10-20 min; the fluorescence intensity was measured, and the amount of decrease in substrate or the amount of carboxyl product produced by the specific fluorescent probe was used as an index for evaluating carboxylesterase 1 activity.
Or adding a fluorogenic probe substrate for measuring the specificity of carboxylesterase 1 into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of an incubation system is 5.5-10.5, and the reaction is carried out for 10-20 min; detecting a specific fluorescent probe substrate or a carboxyl product by using a liquid phase at 256-365nm of ultraviolet, and taking the reduced amount of the specific fluorescent probe substrate or the generated amount of the carboxyl product as an evaluation index of carboxylesterase 1 activity.
The specific fluorescent probe substrate of the invention directly reacts with luciferase, and carboxyl products thereof can be metabolized and oxidized by the luciferase to convert chemical energy into light energy, and a bio-luminescent signal is detected by using an enzyme-labeled instrument, and the full-wavelength scanning is performed; the carboxyl products of the probe substrates and their esters after hydrolysis can also be detected directly by liquid phase, e.g.HPLC or LCMS, with signal values at 256-365nm in the UV.
The method can measure the decrease of probe substrate or the generation of carboxyl product in unit time as the evaluation index of carboxylesterase 1 activity.
Preferably, the reaction temperature is 34 to 42 ℃, more preferably 37 ℃; the pH of the incubation system is 5.5 to 7.0, more preferably ph=6.5.
The sample to be detected is a biological sample, and is any one of recombinant single enzyme containing CES1, human or animal tissue preparation liquid, various mammal tissue cells and preparations thereof.
The probe substrate can also be used for quantitatively detecting CES1 enzyme activity in body fluid, cell or tissue samples from different species. Or can be used for CES1 detection of experimental animals to evaluate individual and species differences of metabolic enzyme CES1.
The probe substrate has high specificity and sensitivity, and can be used for screening and evaluating CES1 inhibitors or; in particular to rapid screening and quantitative evaluation of inhibition capacity of CES1 inhibitors.
The specific fluorogenic probe substrates of the present invention can also be used for fluorescent bioimaging, in particular for fluorescent bioimaging to localize carboxylesterase 1CES1 in biological tissue.
The bioluminescence fluorescence probe substrate and the carboxyl ester bond hydrolysate of CES1 provided by the invention have no spontaneous bioluminescence property, do not interfere with detection, and can realize rapid and sensitive detection of the product by adopting a bioluminescence detector; and (3) detecting the carboxyl ester bond cleavage product by full-wavelength scanning after secondary reaction with luciferase. In addition, the CES1 activity detection process is not easily interfered by biological system matrixes and impurities, and can be used for quantitative determination of CES1 enzyme activity in various recombinant CES1, human and animal tissue preparation solutions and various tissue cells; meanwhile, the probe substrate of the animal integral CES1 can be used for evaluating individual and species differences of the metabolic enzyme CES1. The chemiluminescence detection method after the probe carboxyl product reacts with luciferase secondarily can also be used for rapid screening of CES1 inhibitors and quantitative evaluation of inhibition capacity.
As a fluorescent probe substrate of CES1 single enzyme with high specificity, the compound can be used for detecting the activity of CES1, and is particularly suitable for measuring the enzyme activity of CES1 produced by bacterial, insect, mammalian cell and microzyme clone expression system, and calibrating the activity of CES1 in preparations of microsomes, S9 and the like of various mammalian tissue and organ sources.
The invention has the beneficial effects that the specific fluorescent probe substrate has the following characteristics:
(1) High specificity: can be metabolized into a metabolite, namely a carboxyl ester bond cleavage product, with high specificity by CES1, thereby greatly improving the reliability of the results in biochemical index detection.
(2) The sensitivity is high: quantitative determination of CES1 was performed by the establishment of a ratio-based standard curve with a lower detection limit of 5ng/mL.
(3) The preparation method is simple and easy to obtain: can be obtained by a chemical synthesis method, and the synthesis process is simple and feasible.
(4) Can realize high flux detection and has wide application: the method can be used for measuring various fluorescent enzyme-labeled instruments and biochemicals which are common in laboratories, and can be used for batch detection by using 96 or 386 microplates; in addition to measuring enzyme activity, fluorescence biological imaging can be achieved to locate CES1 in biological tissue, and to screen or evaluate inhibitors or inducers of CES1.
Drawings
FIG. 1 shows a DDM 1 H-NMR spectrum;
FIG. 2 is a DDM 13 C-NMR spectrum;
FIG. 3 is a DDM2 1 H-NMR spectrum;
FIG. 4 is a diagram of DDM2 13 C-NMR spectrum;
FIG. 5 is a selectivity of DDM;
FIG. 6 is a linear reaction time profile of CES1 catalyzed DDM hydrolysis;
FIG. 7 is a quantitative standard curve for CES 1;
FIG. 8 is a graph of the enzymatic kinetics of CES1 catalyzed DDM hydrolysis;
FIG. 9 is a graph of quantitative assessment of CES1 activity in human tissue microsomes;
fig. 10 is CES1 inhibitor screen:
FIG. 10 (a) is a graph showing the result of the inhibition of betulinic acid on the probe substrate (DDM),
FIG. 10 (b) is a graph showing the result of inhibition of probe substrate (DDM) by epi-betulinic acid,
FIG. 10 (c) is a graph showing the result of inhibition of probe substrate (DDM) by liquidambar acid,
FIG. 10 (d) is a graph showing the result of inhibition of probe substrate (DDM) by oleanolic acid;
FIG. 11 is CES1 inhibitor screen:
FIG. 11 (a) is a graph showing the result of the inhibition of betulinic acid on the probe substrate (DDM 2),
FIG. 11 (b) is a graph showing the result of the inhibition of the probe substrate (DDM 2) by epi-betulinic acid,
FIG. 11 (c) is a graph showing the result of inhibition of probe substrate (DDM 2) by liquidambar acid,
FIG. 11 (d) is a graph showing the result of the inhibition of probe substrate (DDM 2) by oleanolic acid;
FIG. 12 measurement of residual activity of liver CES1 following ANIT induced rat bile stasis type liver injury;
FIG. 13 measurement of serum CES1 activity following ANIT induced rat bile stasis type liver injury;
FIG. 14 DDM was used for transfection of LUC + CES1 imaging of different cells and different numbers of cells;
FIG. 15 DME and DDM comparison for transfection of LUC + Mice were imaged for whole body CES1, with DME probe on the left and DDM probe on the right.
Detailed Description
The following examples further illustrate the invention, but are not intended to limit it.
The equipment used in the invention has the following model: the fluorescence emission/excitation spectrum is detected by a synergy H1 full-function micro-pore plate detector; 1 the H-NMR spectrum was carried out by nuclear magnetic resonance spectroscopy (Avance II 400 MHz).
The structural general formula of the bioluminescence probe is:
example 1
Synthesis of DDM
The synthetic route of DDM is as follows:
1) Synthesis of Compound (2):
100mg (0.57 mmol,1 eq) of compound (1) was put into a 100mL reaction flask, 10mL of DMSO was added for dissolution, then 55.49mg of NaCN (1.14 mmol,2 eq) was added, the reaction was stirred for 3-4 hours at 130℃in an oil bath, and TLC detection was performed, the developing solvent being petroleum ether: ethyl acetate=5:1, after completion of the reaction NaHCO was used 3 Adjusting pH of the reaction solution to 7-8, adding ethyl acetate and water for extraction, extracting water phase with ethyl acetate for 2 times (30 mL×2), washing organic phase with water for 1 time (30 mL), washing saturated salt with water for 1 time (30 mL), and anhydrous Na 2 SO 4 Drying (30 min), concentrating by rotary evaporation, and purifying with silica gel column as eluent: ethyl acetate = 5:1, the yellow solid compound is compound (2) with a yield of 30-40%.
2) Synthesis of compound (3):
55.82mg (0.28 mmol,1 eq) of compound (2) was put into a 100mL reaction flask, 10mL of methanol was added for dissolution, 67.85mg of D-cysteine (0.56 mmol,2 eq) was then added, 3mL of water was added, 44.52mg of Na 2 CO 3 (0.42 mmol,1.5 eq) and stirred at ambient temperature for 45min to 1 h, TLC detection, developing solvent petroleum ether: ethyl acetate=5:1, after the reaction, ph=8-9 was adjusted, petroleum ether was used: ethyl acetate = 5:1 with water, removing impurities, adjusting the pH of the aqueous phase to 5-6, extracting the product with ethyl acetate (30 mL,20mL,10 mL), and then with anhydrous Na 2 SO 4 Drying (30 min), and rotary steaming and drying to obtain compound (3) with yield of 50-60%.
3) Synthesis of Compound (4): 40mg (0.13 mmol,1 eq) of compound (3) was charged into a 100mL reaction flask, and 10mL CH was added 2 Cl 2 Then 10mL (1.3 mmol,10 eq) of methanol was added, 4.79mg of EDCI (0.03 mmol,0.2 eq) was added, followed by 31.76mg of DMAP (0).26mmol,2 eq), TLC detection, developing agent petroleum ether ethyl acetate=10:1, after the reaction, extracting with ethyl acetate and water, ethyl acetate extracting water phase 2-3 times, saturated saline water washing 1 time (30 mL), anhydrous Na 2 SO 4 Drying (30 min), concentrating by rotary evaporation, and purifying with silica gel column as eluent: ethyl acetate = 10:1 to obtain a reddish brown compound (4) with a yield of 30-40% and DDM 1 The H-NMR spectrum is shown in figure 1, 13 the C-NMR spectrum is shown in FIG. 2.
Synthesis of DDM2
The synthetic route of DDM2 is as follows:
synthesis of Compound 2-b: 120mg (0.57 mmol,1 eq) of Compound 1-b are taken up in 10ml of dichloromethane, 62.95. Mu.L (1.71 mmol,3 eq) of formaldehyde are added, followed by 211.94mg (1.71 mmol,3 eq) of sodium borohydride acetate, and reacted at room temperature for about 45min, TLC detection, developing agent: ethyl acetate = 10:1, after the reaction was completed, dichloromethane (30 mL. Times.2) and water (20 mL. Times.2) were added to extract twice, and saturated brine was washed once (20 mL. Times.1), anhydrous Na 2 SO 4 Drying (30 min), loading on silica gel column with 200 mesh, eluting with petroleum ether: ethyl acetate = 10:1, the product is yellow green oily matter, and the yield is 85-95%.
Synthesis of Compound 3-b: 63mg (0.28 mmol,1 eq) of Compound 2-b are dissolved in 10ml of DMSO, 27.48mg (0.56 mmol,2 eq) of NaCN are added, a catalytic amount of NaI is added, the reaction is carried out in an oil bath at 130℃for about 4 hours, TLC detection is carried out, the developing agent is petroleum ether: ethyl acetate = 5:1, ending the reaction when the reaction product no longer increases, using NaHCO 3 Adjusting pH of the reaction solution to 7-8, adding ethyl acetate and water for extraction, extracting water phase with ethyl acetate for 2 times (30 mL×2), washing organic phase with water for 1 time (30 mL), washing saturated salt with water for 1 time (30 mL), and anhydrous Na 2 SO 4 Drying (30 min), concentrating by rotary evaporation, and purifying with silica gel column as eluent: ethyl acetate = 5:1, the yellow solid compound is compound 3-b, and the yield is 30-40%.
Synthesis of Compound 4-b: 24mg (0.11 mmol,1 eq) of compound (3) are dissolved in 10ml of dichloromethane, followed by 27.02mg (0.22 mmol,2 eq) of D-Cysteine,17.65mg (0.1665 mmol,1.5 eq) of Na 2 CO 3 And 10mL of water, stirring and reacting for about 1 hour at normal temperature, detecting by TLC, wherein the developing agent is petroleum ether: ethyl acetate = 5:1, after the reaction, ph=8-9 was adjusted, using petroleum ether: ethyl acetate = 5:1 with water, removing impurities, adjusting the pH of the aqueous phase to 5-6, extracting the product with ethyl acetate (30 mL,20mL,10 mL), and then with anhydrous Na 2 SO 4 Drying (30 min), and rotary steaming and drying to obtain compound 4-b with 35-45% yield.
Synthesis of Compound 5-b 14mg (0.044 mmol,1 eq) of Compound 4-b was dissolved in 5mL of dichloromethane, then 4mL (0.44 mmol,10 eq) of methanol was added, 16.82mg of EDCI (0.088 mmol,2 eq) was added, then 11mg of DMAP (0.09 mmol,2 eq) was added, and the reaction was carried out at room temperature for 2 hours, as determined by TLC, with developing solvent of Petroleum ether: ethyl acetate=10:1, after completion of the reaction, extraction was carried out with ethyl acetate and water, extraction of the aqueous phase was carried out 2 to 3 times with ethyl acetate, 1 time with saturated saline (30 mL) and anhydrous Na 2 SO 4 Drying (30 min), concentrating by rotary evaporation, and separating with silica gel column with petroleum ether/ethyl acetate=10:1 as eluent to obtain reddish brown compound 5-b (DMM-2) with 40-50% yield, and DDM2 1 The H-NMR spectrum is shown in figure 3, 13 the C-NMR spectrum is shown in FIG. 4.
Example 2 in vitro determination of Single enzyme Selectivity of human recombinant CES1
(1) The following zymogens were used in the single enzyme screen: catechol-O-methyltransferase (COMT), human Serum Albumin (HSA), alpha-chymotrypsin, lysozyme (Lysaozyme), human carboxylesterase 1 (CES 1A), human carboxylesterase 2 (CES 2A), acetylcholinesterase (Ache), butyrylcholinesterase (Bche), carbonic Anhydrase (CA), human Pancreatic Lipase (HPL), thrombin, alpha-glucosidase, alpha-Amylase (alpha-Amylase), porcine Pancreatic Lipase (PPL), pepsin, pancreatin. The same amount of enzyme was used for the reaction system, which consisted essentially of 5. Mu.L of enzyme (final concentration 10. Mu.g/mL), 2. Mu.L of DDM probe substrate (final concentration 10. Mu.M), 93. Mu.LPBS buffer (pH=6.5). The reaction was initiated by first adding 5. Mu.L of each of the different singleases to 93. Mu.L of PBS buffer, incubating at 37℃for 3 minutes, and then adding 2. Mu.L of DDM probe substrate.
(2) Mixing 50 μl of the reaction solution with 50 μl of Luciferase (LDR), detecting chemiluminescence signals by using a vertical Ma Fangru enzyme-labeled instrument, scanning the whole wave for 1min, detecting for 30min, and comparing the highest value. Each test set up 3 parallel tests, and the mean value was taken for calculation. The probe is specifically hydrolyzed only by recombinant human CES1 single enzyme, and other single enzymes have almost no hydrolysis reaction. The selectivity of DDM is shown in FIG. 5, and it can be seen that the substrate has very high activity selectivity for human carboxylesterase 1.
EXAMPLE 3CES1 time standard curve determination
The reaction system consisted essentially of 2.5. Mu.L CES1 single enzyme (final concentration 0.5. Mu.g/mL), 2. Mu.L DDM probe substrate (final concentration 1.5. Mu.M), 45.5. Mu.L PBS buffer (0.1M, pH=6.5). The reaction process is as follows: firstly, adding 2.5 mu L of CES1 single enzyme into 45.5 mu L of PBS buffer solution, mixing with 50 mu L of Luciferase (LDR), incubating for 3min at 37 ℃, then adding 2 mu L of DDM probe substrate to initiate reaction, detecting a luminescence signal by a vertical Ma Fangru enzyme-labeled instrument, scanning the whole wavelength, detecting once in 1min, continuously detecting for 40min, taking the highest value when the reactions are parallel as a comparison, measuring a metabolite generation amount-time curve, and determining a linear reaction time range. Each experiment was run in parallel with 3 sets of linear reaction time curves for CES1 to catalyze DDM hydrolysis as shown in figure 6.
Example 4 in vitro determination of lower detection limit of CES1
The reaction system mainly comprises 5. Mu.L of enzyme (final concentration was 0.005, 0.010, 0.020, 0.050, 0.100, 0.200, 0.500, 1.000, 2.000, 4.000, 8.000, 16.000, 20.000. Mu.g/mL, respectively), 2. Mu.L of DDM probe substrate (final concentration 10. Mu.M), 93. Mu.L of PBS buffer (0.1M, pH=6.5). The reaction process is that firstly, 5 mu L of enzyme is added into 93 mu L of PBS buffer solution, incubation is carried out for 3min at 37 ℃, then 2 mu L of DDM probe substrate is added for initiating reaction, reaction is carried out for 20min, 50 mu L of reaction solution is mixed with 50 mu L of Luciferase (LDR), a Ma Fangru enzyme label instrument is used for detecting luminescence signals, detection is carried out once for 1min, detection is carried out for 30min, the highest value is taken as comparison, metabolite generation amount-time curve is measured, and the linear reaction enzyme concentration range is determined. Each experiment was set up with 3 sets of parallelism. Each group ofThe average value of (C) was compared with the control group without CES1, and the result showed that the average value was statistically significant at 0.005. Mu.g/mL (R 2 =0.9982, p < 0.0001), CES1 is shown in fig. 7, thus determining a lower limit of detection of CES1 of 5ng/mL in vitro.
EXAMPLE 5CES1 enzymatic kinetic test
Experiments were performed on a microplate reader using 96-well plates, substrate 1-400. Mu.M, CES1 single enzyme 0.02mg/mL, PBS buffer at pH 6.5 100mM, total volume 100. Mu.L, incubated at 37℃for 3min, and analyzed by the microplate reader for 30min every 1 min. Detection conditions: scanning at full wavelength. Substituting the obtained fluorescence intensity into a standard curve to obtain the V of Human Liver Microsome (HLM) to DDM max And K m The enzymatic kinetics of the hydrolysis of DDM catalyzed by CES1 is shown in FIG. 8.
EXAMPLE 6 quantitative detection of CES1 Activity in human liver microsomes
Experiments were performed on a microplate reader using 96-well plates, and the reaction system mainly comprises 2 μl (4 μΜ, final concentration) of DDM probe substrate, 5 μl (final concentrations of 0.5, 1, 2.5, 5, 10, 20, 50, 100, 200, 400 μg/mL respectively) of CES1 single enzyme, 93 μl PBS buffer (0.1M, ph=6.5), and the reaction process is: firstly, adding 5 mu L of enzyme into 93 mu L of PBS buffer solution, incubating for 3min at 37 ℃, then adding 2 mu L of DDM probe substrate to initiate reaction for 20min, mixing 50 mu L of reaction solution with 50 mu L of Luciferase (LDR), detecting a luminescence signal by a vertical Ma Fangru enzyme-labeled instrument, detecting once for 1min, continuously detecting for 30min, taking the highest value as comparison, determining a metabolite generation amount-time curve, determining an enzyme activity linear reaction range, and quantitatively evaluating the activity of CES1 in human tissue microsomes, wherein the activity quantitative evaluation curve is shown in figure 9.
EXAMPLE 7CES1 inhibitor Primary screening
The inhibition of CES1 mediated hydrolysis of two probes was tested by selecting 4 natural compounds, the final concentration of the inhibitor was set to 1. Mu.M, 10. Mu.M, 100. Mu.M, the final concentration of the two probe substrates was 2. Mu.M, the final concentration of the enzyme was 1. Mu.g/mL, the assay was performed on a microplate reader using 96-well plates, the enzyme, inhibitors at each concentration and buffer were pre-incubated at 37℃for 3min, then probe substrates were added to initiate the reaction, 50. Mu.L of the reaction solution was taken after 10min, 50. Mu.L of luciferase detection reagent was added, and then the reaction solution was immediately placed into the microplate reader to be detected for 30min, once every 1 min. Detection conditions: scanning at full wavelength. Each set of experiments set up 3 data in parallel. CES1 inhibitor screening is shown in figures 10 and 11, wherein figure 10 (a) is a diagram showing the result of the inhibition of betulinic acid on probe substrate (DDM), figure 10 (b) is a diagram showing the result of the inhibition of table-betulinic acid on probe substrate (DDM), figure 10 (c) is a diagram showing the result of the inhibition of lupulic acid on probe substrate (DDM), and figure 10 (d) is a diagram showing the result of the inhibition of oleanolic acid on probe substrate (DDM);
FIG. 11 (a) is a graph showing the result of the inhibition of betulinic acid on the probe substrate (DDM 2), FIG. 11 (b) is a graph showing the result of the inhibition of table-betulinic acid on the probe substrate (DDM 2), FIG. 11 (c) is a graph showing the result of the inhibition of lupulic acid on the probe substrate (DDM 2), and FIG. 11 (d) is a graph showing the result of the inhibition of oleanolic acid on the probe substrate (DDM 2).
Example 8 animal tissue carboxylesterase 1 (CES 1) residual enzyme Activity assay
S9 preparation of homogenate preparation tissue S9: weighing the tissue, shearing the tissue, adding 5 times of PBS (phosphate buffered saline), preparing tissue homogenate in a low-temperature homogenate medium, centrifuging for 20min at 4 ℃ by 9000g, taking the supernatant as the tissue S9, and quick-freezing the tissue S9 at-80 ℃ for later detection.
Assay of carboxylesterase 1 (CES 1) serum frozen at-80 ℃ and prepared liver tissue S9 were taken, the activity level of CES1 was detected, and the specific probe substrate NLMe for CES1 assay was independently developed using the present laboratory.
Preparation: PBS phosphate buffer with pH of 6.5, CES1 probe substrate NLMe (final concentration 2. Mu.M), reaction temperature of 37℃and the generation system is as follows:
1) The CES1 total reaction concentration system (volume 100 μl) included: 2. Mu.L of substrate (NLMe), 5. Mu.L of tissue S9, 93. Mu.L of PBS phosphate buffer;
2) Adding luciferase to start luminescence reaction, wherein the reaction time is 10min;
3) And (3) placing the sample into an enzyme-labeled instrument, selecting full-wave detection, and measuring the hydrolysis yield of lactone bonds in unit time as an evaluation index of CES1 activity.
The whole study was performed in triplicate, and the measurement results of residual activity of liver CES1 after ANIT induced rat gall stasis type liver injury are shown in FIG. 12 and FIG. 13, respectively.
EXAMPLE 9DDM was used to transfect LUC + CES1 imaging of different cells and different numbers of cells
3 different cells are selected to test CES1 imaging effect of DDM on a cell level, wherein the HepG2 cells are human liver cancer cells, the HCT116 cells are human colon cancer cells, the U87 cells are human brain astrocyte tumor cells, and the three cells are transfected with a Luciferase reporter gene. According to the principle of probe luminescence, when CES1 is present in the cell, DDM is hydrolyzed by CES1 into a carboxyl product, and then undergoes oxidation reaction with luciferase (Luciferin) to release fluorescence.
All three cells used 5X 10 5 、2.5×10 5 、1×10 5 Three different numbers, we can observe the number-dependent imaging brightness, as a comparison, we made a list of 5×10 5 Control experiment of quantitative cells plus CES1 Positive inhibitor BNPP DDM was used to transfect LUC as shown in FIG. 14 + The experimental results of CES1 imaging of different cells and different numbers of cells show that the human liver cancer cells have the largest CES1, the colon cancer cells are inferior, almost no CES1 exists in brain astrocytoma cells, and positive inhibitors inhibit CES1 at the cell level.
EXAMPLE 10DDM used for transfection of LUC + Whole body CES1 imaging in mice
The mice transfected with the Luciferase reporter gene in a whole body are selected, and DDM and the previous generation probe DME are injected into the mice simultaneously for imaging and comparison. The mice on the left side were intraperitoneally injected with 0.1mL of 5mM DME probe, the mice on the right side were intraperitoneally injected with 0.1mL of 5mM DDM probe, and after the injection was completed, both mice were simultaneously observed by imaging, and the contrast of DME and DDM was used for imaging the whole body CES1 of the transfected LUC+ mice, as shown in FIG. 15, with DME probe on the left side and DDM probe on the right side.
Discussion:
according to the results of the embodiment, the application of the bioluminescence fluorescence probe reaction of CES1 provided by the invention can be seen, the probe substrate and the carboxyl ester bond hydrolysate have no spontaneous bioluminescence property, the detection cannot be interfered, and the bioluminescence detector can be adopted to realize the rapid and sensitive detection of the product. In addition, the method is not easily interfered by biological system matrixes and impurities in the CES1 activity detection process, can be used for quantitative determination of CES1 enzyme activity in various recombinant CES1, human and animal tissue preparation solutions and various tissue cells, and can also be used for rapid screening of CES1 inhibitors and quantitative evaluation of inhibition capability; meanwhile, the probe substrate of the animal integral CES1 can be used for evaluating individual and species differences of the metabolic enzyme CES1.
Furthermore, as a fluorescent probe substrate of CES1 single enzyme with high specificity, the compound can be used for detecting the activity of CES1, and is particularly suitable for measuring the enzyme activity of CES1 produced by bacterial, insect, mammalian cells and saccharomycete clone expression systems, and calibrating the activity of CES1 in preparations of microsomes, S9 and the like derived from various mammalian tissues and organs.
Regardless of the application, the CES1 fluorogenic probe substrate of the present invention has high specificity for detecting CES1 single enzyme in vitro activity, and in embodiments DDM can be metabolized to a metabolite with high specificity by CES 1; the method has the advantages of easy high-flux detection, can be measured on various fluorescent enzyme-labeled instruments and biochemical instruments which are common in laboratories in embodiments, and can be used for batch detection by using 96 or 386 microplates; and the detection result also shows that the detection method has high sensitivity, the quantitative determination of CES1 can be carried out through the establishment of a ratio type standard curve, and the detection lower limit can reach 5ng/mL. Therefore, the CES1 fluorescent probe substrate has high specificity, high sensitivity, high detection convenience and high flux detection performance in the in-vitro activity of CES1 single enzyme, has different detection modes compared with a fluorescent probe, is basically not interfered by biological matrixes, and has higher selectivity and accuracy.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any changes or substitutions that do not undergo the inventive effort should be construed as falling within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope defined by the claims.
Claims (8)
1. A specific fluorogenic probe substrate for measuring carboxylesterase 1, characterized by the following structural formula:
wherein R is 1 Is H or methyl, R 2 Is methyl.
2. The method for preparing a specific fluorogenic probe substrate for assaying carboxylesterase 1 according to claim 1, comprising the steps of:
1) Performing cyano substitution reaction on the compound 2 to obtain a compound 3;
2) Compound 3 undergoes cyano hydrolysis to the corresponding acid;
3) The specific fluorogenic probe substrate for measuring carboxylesterase 1 is generated through esterification reaction.
3. Use of a specific fluorogenic probe substrate for assaying carboxylesterase 1 according to claim 1 for the preparation of a reagent for assaying carboxylesterase 1 or for assaying carboxylesterase 1 activity.
4. A method for measuring carboxylesterase 1 activity for non-diagnostic purposes, which is characterized in that a specific fluorogenic probe substrate according to claim 1 is reacted with a sample to be measured to measure the carboxylesterase 1, either qualitatively or quantitatively, of a specific fluorogenic probe substrate or of a hydrolyzed carboxyl product thereof.
5. The method of claim 4, wherein the steps include:
adding a fluorogenic probe substrate for measuring the specificity of carboxylesterase 1 into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of an incubation system is 5.5-10.5, and adding luciferase to start bioluminescence reaction, wherein the reaction time is 10-20 min; detecting fluorescence intensity, and taking the reduced amount of the substrate of the specific fluorescent probe or the generated amount of carboxyl product as an evaluation index of carboxylesterase 1 activity;
or adding a fluorogenic probe substrate for measuring the specificity of carboxylesterase 1 into a phosphate buffer system, wherein the reaction temperature is 20-60 ℃, the pH of an incubation system is 5.5-10.5, and the reaction is carried out for 10-20 min; detecting a specific fluorescent probe substrate or a carboxyl product by using a liquid phase at 256-365nm of ultraviolet, and taking the reduced amount of the specific fluorescent probe substrate or the generated amount of the carboxyl product as an evaluation index of carboxylesterase 1 activity.
6. The method according to claim 5, wherein the reaction temperature is 34-42℃and the pH of the incubation system is 5.5-7.0.
7. The method of claim 5, wherein the specific fluorogenic probe substrate or carboxyl product is detected by HPLC or LCMS at ultraviolet 256-365 nm.
8. Use of a specific fluorogenic probe substrate for assaying carboxylesterase 1 according to claim 1 for in vitro screening of inhibitors or inducers of carboxylesterase 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111597847.9A CN114478578B (en) | 2021-12-24 | 2021-12-24 | Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111597847.9A CN114478578B (en) | 2021-12-24 | 2021-12-24 | Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114478578A CN114478578A (en) | 2022-05-13 |
| CN114478578B true CN114478578B (en) | 2024-03-29 |
Family
ID=81496367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111597847.9A Active CN114478578B (en) | 2021-12-24 | 2021-12-24 | Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114478578B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105712987A (en) * | 2016-03-03 | 2016-06-29 | 中国科学院大连化学物理研究所 | Bioluminescence probe substrate for human carboxylesterase 1 and preparation method and application thereof |
| CN106546577A (en) * | 2015-09-21 | 2017-03-29 | 中国科学院大连化学物理研究所 | The bioluminescent assay kit and its using method of human carboxylatase 1 and application |
| CN107271432A (en) * | 2016-04-08 | 2017-10-20 | 中国科学院大连化学物理研究所 | The bioluminescent assay kit and its application method of human carboxylatase 1 and application |
| CN111675724A (en) * | 2020-07-21 | 2020-09-18 | 山东大学 | A kind of luciferase substrate and its preparation method and application |
| CN112778435A (en) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | Method for separating and purifying dendrobium officinale polysaccharide by aqueous two-phase extraction |
| CN113004432A (en) * | 2021-03-05 | 2021-06-22 | 中国科学院昆明植物研究所 | Dendrobium officinale oligosaccharide, dendrobium officinale oligosaccharide derivative and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7104702B2 (en) * | 2016-12-01 | 2022-07-21 | プロメガ コーポレイション | 5,5-Disubstituted luciferin and their use in luciferase based assays |
-
2021
- 2021-12-24 CN CN202111597847.9A patent/CN114478578B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546577A (en) * | 2015-09-21 | 2017-03-29 | 中国科学院大连化学物理研究所 | The bioluminescent assay kit and its using method of human carboxylatase 1 and application |
| CN105712987A (en) * | 2016-03-03 | 2016-06-29 | 中国科学院大连化学物理研究所 | Bioluminescence probe substrate for human carboxylesterase 1 and preparation method and application thereof |
| CN107271432A (en) * | 2016-04-08 | 2017-10-20 | 中国科学院大连化学物理研究所 | The bioluminescent assay kit and its application method of human carboxylatase 1 and application |
| CN111675724A (en) * | 2020-07-21 | 2020-09-18 | 山东大学 | A kind of luciferase substrate and its preparation method and application |
| CN113004432A (en) * | 2021-03-05 | 2021-06-22 | 中国科学院昆明植物研究所 | Dendrobium officinale oligosaccharide, dendrobium officinale oligosaccharide derivative and preparation method and application thereof |
| CN112778435A (en) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | Method for separating and purifying dendrobium officinale polysaccharide by aqueous two-phase extraction |
Non-Patent Citations (4)
| Title |
|---|
| A synthetic luciferin improves bioluminescence imaging in live mice;Melanie S. Evans 等;《Nat Methods》;第11卷(第4期);第393-395页，尤其是文章摘要以及图1 * |
| Aminoluciferins Extend Firefly Luciferase Bioluminescence into the Near-Infrared and Can Be Preferred Substrates over D‑Luciferin;David M. Mofford 等;《J. Am. Chem. Soc》;第136卷;第13277-13282页 * |
| David M. Mofford 等.Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.《J. Am. Chem. Soc.》.2015,第137卷(第27期),第8684-8687页，尤其是附加信息第S11页化合物16，第8684页图1，第8685页右栏倒数第1段. * |
| Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity;David M. Mofford 等;《J. Am. Chem. Soc.》;第137卷(第27期);第8684-8687页，尤其是附加信息第S11页化合物16 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114478578A (en) | 2022-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | A bioluminescent sensor for highly selective and sensitive detection of human carboxylesterase 1 in complex biological samples | |
| Lippert et al. | A hydrogen peroxide-responsive hyperpolarized 13C MRI contrast agent | |
| Liu et al. | A ratiometric fluorescent sensor for highly selective detection of human carboxylesterase 2 and its application in living cells | |
| Lan et al. | Detection techniques of carboxylesterase activity: An update review | |
| JP6281727B2 (en) | Specific fluorescent probe and quantitative method based on pseudoesterase hydrolysis reaction of albumin | |
| Ma et al. | Rational design of a near-infrared fluorescence probe for highly selective sensing butyrylcholinesterase (BChE) and its bioimaging applications in living cell | |
| Xiang et al. | Ratiometric imaging of butyrylcholinesterase activity in mice with nonalcoholic fatty liver using an AIE-based fluorescent probe | |
| Ge et al. | A novel substrate-inspired fluorescent probe to monitor native albumin in human plasma and living cells | |
| Mizukami et al. | 19 F MRI detection of β-galactosidase activity for imaging of gene expression | |
| Wang et al. | Turn-on visible and ratiometric near-infrared fluorescent probes for distinction endogenous esterases and chymotrypsins in live cells | |
| Liu et al. | New fluorescent probe with recognition moiety of bipiperidinyl reveals the rise of hepatocellular carboxylesterase activity during heat shock | |
| Nilam et al. | Enzyme assays with supramolecular chemosensors–the label-free approach | |
| CN106841128B (en) | Application of high-specificity fluorescent probe for detecting human serum albumin | |
| Wang et al. | A chemiluminescent probe for the real-time monitoring of esterases activities | |
| Zhang et al. | A far-red/near-infrared fluorescence probe with large Stokes shift for monitoring butyrylcholinesterase (BChE) in living cells and in vivo | |
| Liu et al. | Selective and sensitive detection and quantification of human carboxylesterase 1 by a ratiometric fluorescence probe | |
| Li et al. | Screening of lactate dehydrogenase inhibitor from bioactive compounds in natural products by electrophoretically mediated microanalysis | |
| Wang et al. | Sensing and imaging of exosomal CD26 secreted from cancer cells and 3D colorectal tumor model using a novel near-infrared fluorogenic probe | |
| CN114478578B (en) | Specific bioluminescence probe substrate for measuring carboxylesterase 1 and preparation method and application thereof | |
| Acari et al. | Real-time visualization of butyrylcholinesterase activity using a highly selective and sensitive chemiluminescent probe | |
| Sun et al. | Coumarin-based turn-on fluorescence probe with a large Stokes shift for detection of endogenous neutrophil elastase in live cells and zebrafish | |
| Wang et al. | A red emission multiple detection site probe for detecting carboxylesterase 1 based on BODIPY fluorophore | |
| Wang et al. | Design and synthesis of a new near-infrared and large Stokes shift fluorescence probe for NAD (P) H: quinone oxidoreductase 1 detection in living systems | |
| CN106546577B (en) | The bioluminescent assay kit and its application method of human carboxylatase 1 and application | |
| CN118598926A (en) | Self-fixed near-infrared small molecule fluorescent probe and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |