EP1026988A1 - Novel fluorescent reporter molecules and their applications including assays for caspases - Google Patents
Novel fluorescent reporter molecules and their applications including assays for caspasesInfo
- Publication number
- EP1026988A1 EP1026988A1 EP98953317A EP98953317A EP1026988A1 EP 1026988 A1 EP1026988 A1 EP 1026988A1 EP 98953317 A EP98953317 A EP 98953317A EP 98953317 A EP98953317 A EP 98953317A EP 1026988 A1 EP1026988 A1 EP 1026988A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- rhodamine
- cells
- compound
- asp
- 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.)
- Withdrawn
Links
- 102000011727 Caspases Human genes 0.000 title claims abstract description 119
- 108010076667 Caspases Proteins 0.000 title claims abstract description 119
- 238000003556 assay Methods 0.000 title abstract description 85
- 150000001875 compounds Chemical class 0.000 claims abstract description 367
- 102000035195 Peptidases Human genes 0.000 claims abstract description 135
- 108091005804 Peptidases Proteins 0.000 claims abstract description 135
- 238000000034 method Methods 0.000 claims abstract description 113
- 239000004365 Protease Substances 0.000 claims abstract description 96
- 102000004190 Enzymes Human genes 0.000 claims abstract description 86
- 108090000790 Enzymes Proteins 0.000 claims abstract description 86
- 235000019419 proteases Nutrition 0.000 claims abstract description 82
- 230000000694 effects Effects 0.000 claims abstract description 65
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 62
- 230000006907 apoptotic process Effects 0.000 claims abstract description 60
- 201000011510 cancer Diseases 0.000 claims abstract description 47
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 43
- 108010010369 HIV Protease Proteins 0.000 claims abstract description 31
- 238000011282 treatment Methods 0.000 claims abstract description 28
- 108090000915 Aminopeptidases Proteins 0.000 claims abstract description 24
- 102000004400 Aminopeptidases Human genes 0.000 claims abstract description 24
- 210000000056 organ Anatomy 0.000 claims abstract description 21
- 101710118538 Protease Proteins 0.000 claims abstract description 15
- 101710181812 Methionine aminopeptidase Proteins 0.000 claims abstract description 11
- 108700016158 assemblin Proteins 0.000 claims abstract description 10
- 229940044683 chemotherapy drug Drugs 0.000 claims abstract description 9
- 102000016622 Dipeptidyl Peptidase 4 Human genes 0.000 claims abstract description 5
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 claims abstract description 5
- 102000007590 Calpain Human genes 0.000 claims abstract description 4
- 108010032088 Calpain Proteins 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 469
- 239000000758 substrate Substances 0.000 claims description 153
- 238000012360 testing method Methods 0.000 claims description 150
- 150000001413 amino acids Chemical class 0.000 claims description 136
- 239000000126 substance Substances 0.000 claims description 68
- 229940024606 amino acid Drugs 0.000 claims description 56
- 235000001014 amino acid Nutrition 0.000 claims description 56
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 52
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical group [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 46
- -1 acetoxymethyl Chemical group 0.000 claims description 44
- 230000030833 cell death Effects 0.000 claims description 43
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 42
- 235000019833 protease Nutrition 0.000 claims description 39
- 230000003612 virological effect Effects 0.000 claims description 39
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 36
- 239000001257 hydrogen Substances 0.000 claims description 33
- 229910052739 hydrogen Inorganic materials 0.000 claims description 33
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 31
- JNGRENQDBKMCCR-UHFFFAOYSA-N 2-(3-amino-6-iminoxanthen-9-yl)benzoic acid;hydrochloride Chemical compound [Cl-].C=12C=CC(=[NH2+])C=C2OC2=CC(N)=CC=C2C=1C1=CC=CC=C1C(O)=O JNGRENQDBKMCCR-UHFFFAOYSA-N 0.000 claims description 30
- 210000001519 tissue Anatomy 0.000 claims description 29
- 241001465754 Metazoa Species 0.000 claims description 26
- 230000008859 change Effects 0.000 claims description 26
- 150000003839 salts Chemical class 0.000 claims description 25
- 230000000903 blocking effect Effects 0.000 claims description 22
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims description 21
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 19
- 125000006239 protecting group Chemical group 0.000 claims description 19
- HULKIXRFKRCRHD-UHFFFAOYSA-N 4-[(2-acetamido-4-methylpentanoyl)amino]-5-[[1-[[3-carboxy-1-oxo-1-[[2-oxo-4-(trifluoromethyl)chromen-7-yl]amino]propan-2-yl]amino]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound C=1C=C2C(C(F)(F)F)=CC(=O)OC2=CC=1NC(=O)C(CC(O)=O)NC(=O)C(NC(=O)C(CCC(O)=O)NC(=O)C(NC(C)=O)CC(C)C)CC1=CN=CN1 HULKIXRFKRCRHD-UHFFFAOYSA-N 0.000 claims description 18
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 17
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 claims description 15
- 229940127089 cytotoxic agent Drugs 0.000 claims description 15
- 241000701024 Human betaherpesvirus 5 Species 0.000 claims description 14
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 claims description 14
- 235000008206 alpha-amino acids Nutrition 0.000 claims description 14
- 150000001576 beta-amino acids Chemical class 0.000 claims description 14
- 150000002431 hydrogen Chemical class 0.000 claims description 14
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 10
- 239000008280 blood Substances 0.000 claims description 10
- 150000002148 esters Chemical class 0.000 claims description 10
- 230000003834 intracellular effect Effects 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- AOUOVFRSCMDPFA-QSDJMHMYSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-methylbutanoyl]amino]butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O AOUOVFRSCMDPFA-QSDJMHMYSA-N 0.000 claims description 9
- XVZUMQAMCYSUMS-SIUGBPQLSA-N OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 XVZUMQAMCYSUMS-SIUGBPQLSA-N 0.000 claims description 9
- UPUGLJYNCXXUQV-UHFFFAOYSA-N Oxydisulfoton Chemical compound CCOP(=S)(OCC)SCCS(=O)CC UPUGLJYNCXXUQV-UHFFFAOYSA-N 0.000 claims description 9
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 9
- 102000001398 Granzyme Human genes 0.000 claims description 8
- 108060005986 Granzyme Proteins 0.000 claims description 8
- 210000000987 immune system Anatomy 0.000 claims description 8
- 210000003734 kidney Anatomy 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 102000005962 receptors Human genes 0.000 claims description 8
- 210000004556 brain Anatomy 0.000 claims description 7
- 210000002889 endothelial cell Anatomy 0.000 claims description 7
- 210000002216 heart Anatomy 0.000 claims description 7
- 210000004185 liver Anatomy 0.000 claims description 7
- 125000002801 octanoyl group Chemical group C(CCCCCCC)(=O)* 0.000 claims description 7
- 210000001428 peripheral nervous system Anatomy 0.000 claims description 7
- 210000003491 skin Anatomy 0.000 claims description 7
- 210000004204 blood vessel Anatomy 0.000 claims description 6
- 210000000988 bone and bone Anatomy 0.000 claims description 6
- 210000001185 bone marrow Anatomy 0.000 claims description 6
- 210000003372 endocrine gland Anatomy 0.000 claims description 6
- 210000003238 esophagus Anatomy 0.000 claims description 6
- 210000001508 eye Anatomy 0.000 claims description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 6
- 210000004907 gland Anatomy 0.000 claims description 6
- 210000004072 lung Anatomy 0.000 claims description 6
- 210000001165 lymph node Anatomy 0.000 claims description 6
- 210000000214 mouth Anatomy 0.000 claims description 6
- 210000001331 nose Anatomy 0.000 claims description 6
- 210000002741 palatine tonsil Anatomy 0.000 claims description 6
- 210000000496 pancreas Anatomy 0.000 claims description 6
- 230000001850 reproductive effect Effects 0.000 claims description 6
- 210000000952 spleen Anatomy 0.000 claims description 6
- 210000002784 stomach Anatomy 0.000 claims description 6
- 210000001541 thymus gland Anatomy 0.000 claims description 6
- 210000000515 tooth Anatomy 0.000 claims description 6
- 241000701161 unidentified adenovirus Species 0.000 claims description 6
- 210000003932 urinary bladder Anatomy 0.000 claims description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 5
- 210000001503 joint Anatomy 0.000 claims description 5
- 210000002569 neuron Anatomy 0.000 claims description 5
- 210000004209 hair Anatomy 0.000 claims description 4
- 125000003104 hexanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 4
- 230000035945 sensitivity Effects 0.000 claims description 4
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 3
- 210000002865 immune cell Anatomy 0.000 claims description 3
- 210000004927 skin cell Anatomy 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 241000238631 Hexapoda Species 0.000 claims description 2
- 241000124008 Mammalia Species 0.000 claims description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 125000005907 alkyl ester group Chemical group 0.000 claims description 2
- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000004674 methylcarbonyl group Chemical group CC(=O)* 0.000 claims description 2
- 230000002107 myocardial effect Effects 0.000 claims description 2
- 210000000481 breast Anatomy 0.000 claims 2
- 210000004899 c-terminal region Anatomy 0.000 claims 2
- 102100037119 Mas-related G-protein coupled receptor member G Human genes 0.000 claims 1
- 101150033080 Mrgprg gene Proteins 0.000 claims 1
- 210000002768 hair cell Anatomy 0.000 claims 1
- 238000012544 monitoring process Methods 0.000 claims 1
- 239000003112 inhibitor Substances 0.000 abstract description 48
- 239000007850 fluorescent dye Substances 0.000 abstract description 30
- 241000700588 Human alphaherpesvirus 1 Species 0.000 abstract description 18
- 230000008569 process Effects 0.000 abstract description 16
- 238000001952 enzyme assay Methods 0.000 abstract description 13
- 230000035572 chemosensitivity Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 9
- 239000000411 inducer Substances 0.000 abstract description 5
- 238000007877 drug screening Methods 0.000 abstract description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 93
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 88
- 229940088598 enzyme Drugs 0.000 description 74
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 61
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 46
- 238000003776 cleavage reaction Methods 0.000 description 45
- 230000007017 scission Effects 0.000 description 45
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 42
- 235000019439 ethyl acetate Nutrition 0.000 description 41
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 39
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 38
- 238000012216 screening Methods 0.000 description 36
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 35
- 239000003814 drug Substances 0.000 description 33
- 239000000243 solution Substances 0.000 description 33
- 229940079593 drug Drugs 0.000 description 32
- 239000007787 solid Substances 0.000 description 32
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 28
- 102100029855 Caspase-3 Human genes 0.000 description 27
- 125000000539 amino acid group Chemical group 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 22
- 241000711549 Hepacivirus C Species 0.000 description 20
- 125000000217 alkyl group Chemical group 0.000 description 20
- 238000007423 screening assay Methods 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 19
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 19
- 101000793880 Homo sapiens Caspase-3 Proteins 0.000 description 18
- 150000004702 methyl esters Chemical group 0.000 description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- 241000725303 Human immunodeficiency virus Species 0.000 description 14
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 14
- 230000004913 activation Effects 0.000 description 14
- 125000002252 acyl group Chemical group 0.000 description 14
- 229940041181 antineoplastic drug Drugs 0.000 description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 14
- 230000001413 cellular effect Effects 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 239000002904 solvent Substances 0.000 description 13
- XYXYXSKSTZAEJW-VIFPVBQESA-N (2s)-2-(phenylmethoxycarbonylamino)butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 XYXYXSKSTZAEJW-VIFPVBQESA-N 0.000 description 12
- 108090000397 Caspase 3 Proteins 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 208000031886 HIV Infections Diseases 0.000 description 10
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 description 10
- 230000001640 apoptogenic effect Effects 0.000 description 10
- 239000004030 hiv protease inhibitor Substances 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- 102000004091 Caspase-8 Human genes 0.000 description 9
- 108090000538 Caspase-8 Proteins 0.000 description 9
- 208000037357 HIV infectious disease Diseases 0.000 description 9
- 125000003118 aryl group Chemical group 0.000 description 9
- 230000008901 benefit Effects 0.000 description 9
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 239000013592 cell lysate Substances 0.000 description 8
- 230000034994 death Effects 0.000 description 8
- 239000000975 dye Substances 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- 201000004384 Alopecia Diseases 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- MSHZHSPISPJWHW-PVDLLORBSA-N O-(chloroacetylcarbamoyl)fumagillol Chemical compound C([C@H]([C@H]([C@@H]1[C@]2(C)[C@H](O2)CC=C(C)C)OC)OC(=O)NC(=O)CCl)C[C@@]21CO2 MSHZHSPISPJWHW-PVDLLORBSA-N 0.000 description 7
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 210000004789 organ system Anatomy 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 229960003048 vinblastine Drugs 0.000 description 7
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 6
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 239000012043 crude product Substances 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 230000003676 hair loss Effects 0.000 description 6
- 208000028867 ischemia Diseases 0.000 description 6
- 239000006166 lysate Substances 0.000 description 6
- 102000006240 membrane receptors Human genes 0.000 description 6
- 108020004084 membrane receptors Proteins 0.000 description 6
- 229930182817 methionine Natural products 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 150000001412 amines Chemical class 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
- 238000003271 compound fluorescence assay Methods 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 125000001188 haloalkyl group Chemical group 0.000 description 5
- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 description 5
- 208000015122 neurodegenerative disease Diseases 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 101000983528 Homo sapiens Caspase-8 Proteins 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical compound ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000004037 angiogenesis inhibitor Substances 0.000 description 4
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 4
- 230000001093 anti-cancer Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000002405 diagnostic procedure Methods 0.000 description 4
- 125000004705 ethylthio group Chemical group C(C)S* 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 208000024963 hair loss Diseases 0.000 description 4
- 238000013537 high throughput screening Methods 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 230000035699 permeability Effects 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000002797 proteolythic effect Effects 0.000 description 4
- 230000005855 radiation Effects 0.000 description 4
- 239000001022 rhodamine dye Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- NPDBDJFLKKQMCM-SCSAIBSYSA-N (2s)-2-amino-3,3-dimethylbutanoic acid Chemical compound CC(C)(C)[C@H](N)C(O)=O NPDBDJFLKKQMCM-SCSAIBSYSA-N 0.000 description 3
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 3
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 3
- SKUFPLMENACLEM-UHFFFAOYSA-N 2-(2-ethoxyethoxy)ethoxymethyl carbonochloridate Chemical compound CCOCCOCCOCOC(Cl)=O SKUFPLMENACLEM-UHFFFAOYSA-N 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- UMBVAPCONCILTL-MRHIQRDNSA-N Ac-Asp-Glu-Val-Asp-H Chemical compound OC(=O)C[C@@H](C=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(C)=O UMBVAPCONCILTL-MRHIQRDNSA-N 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 3
- 206010007559 Cardiac failure congestive Diseases 0.000 description 3
- 108090000425 Caspase 6 Proteins 0.000 description 3
- 102000004018 Caspase 6 Human genes 0.000 description 3
- 229940123169 Caspase inhibitor Drugs 0.000 description 3
- 102100038902 Caspase-7 Human genes 0.000 description 3
- 102000005927 Cysteine Proteases Human genes 0.000 description 3
- 108010005843 Cysteine Proteases Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 206010019280 Heart failures Diseases 0.000 description 3
- 101000741014 Homo sapiens Caspase-7 Proteins 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 239000007990 PIPES buffer Substances 0.000 description 3
- 208000017442 Retinal disease Diseases 0.000 description 3
- 102000012479 Serine Proteases Human genes 0.000 description 3
- 108010022999 Serine Proteases Proteins 0.000 description 3
- VNYDHJARLHNEGA-RYUDHWBXSA-N Tyr-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 VNYDHJARLHNEGA-RYUDHWBXSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 108010066665 acetyl-aspartyl-glutamyl-valyl-aspartal Proteins 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 150000003862 amino acid derivatives Chemical class 0.000 description 3
- 125000005140 aralkylsulfonyl group Chemical group 0.000 description 3
- 229960005261 aspartic acid Drugs 0.000 description 3
- 239000012131 assay buffer Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000973 chemotherapeutic effect Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000006482 condensation reaction Methods 0.000 description 3
- 230000003412 degenerative effect Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 125000001165 hydrophobic group Chemical group 0.000 description 3
- 206010020718 hyperplasia Diseases 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 239000006225 natural substrate Substances 0.000 description 3
- 210000000653 nervous system Anatomy 0.000 description 3
- 239000002547 new drug Substances 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 230000002028 premature Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000011269 treatment regimen Methods 0.000 description 3
- 108010020532 tyrosyl-proline Proteins 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- CANZBRDGRHNSGZ-NSHDSACASA-N (2s)-3-methyl-2-(phenylmethoxycarbonylamino)butanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 CANZBRDGRHNSGZ-NSHDSACASA-N 0.000 description 2
- PMBXCGGQNSVESQ-UHFFFAOYSA-N 1-Hexanethiol Chemical compound CCCCCCS PMBXCGGQNSVESQ-UHFFFAOYSA-N 0.000 description 2
- ZTKQHJHANLVEBM-UHFFFAOYSA-N 2-[3-(ethylamino)-6-ethylimino-2,7-dimethylxanthen-9-yl]benzoic acid Chemical compound C1=2C=C(C)C(NCC)=CC=2OC2=CC(=NCC)C(C)=CC2=C1C1=CC=CC=C1C(O)=O ZTKQHJHANLVEBM-UHFFFAOYSA-N 0.000 description 2
- PGRMUEVKABEERE-UHFFFAOYSA-N 2-[3-(methylamino)-6-methyliminoxanthen-9-yl]benzoic acid;perchloric acid Chemical compound [O-]Cl(=O)(=O)=O.C=12C=CC(=[NH+]C)C=C2OC2=CC(NC)=CC=C2C=1C1=CC=CC=C1C(O)=O PGRMUEVKABEERE-UHFFFAOYSA-N 0.000 description 2
- MNKKXTGFPVEFRY-UHFFFAOYSA-N 2-butoxyethyl carbonochloridate Chemical compound CCCCOCCOC(Cl)=O MNKKXTGFPVEFRY-UHFFFAOYSA-N 0.000 description 2
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 description 2
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 102400000344 Angiotensin-1 Human genes 0.000 description 2
- 101800000734 Angiotensin-1 Proteins 0.000 description 2
- HAVKMRGWNXMCDR-STQMWFEESA-N Arg-Gly-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HAVKMRGWNXMCDR-STQMWFEESA-N 0.000 description 2
- 206010003694 Atrophy Diseases 0.000 description 2
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 2
- 108090000567 Caspase 7 Proteins 0.000 description 2
- 102100026548 Caspase-8 Human genes 0.000 description 2
- 102000004225 Cathepsin B Human genes 0.000 description 2
- 108090000712 Cathepsin B Proteins 0.000 description 2
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- YIIMEMSDCNDGTB-UHFFFAOYSA-N Dimethylcarbamoyl chloride Chemical compound CN(C)C(Cl)=O YIIMEMSDCNDGTB-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- 102000018389 Exopeptidases Human genes 0.000 description 2
- 108010091443 Exopeptidases Proteins 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 241001326189 Gyrodactylus prostae Species 0.000 description 2
- 208000004898 Herpes Labialis Diseases 0.000 description 2
- 101000741087 Homo sapiens Caspase-6 Proteins 0.000 description 2
- 208000023105 Huntington disease Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- MDVZJYGNAGLPGJ-KKUMJFAQSA-N Leu-Asn-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MDVZJYGNAGLPGJ-KKUMJFAQSA-N 0.000 description 2
- KFKWRHQBZQICHA-STQMWFEESA-N Leu-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-N 0.000 description 2
- CJUMAFVKTCBCJK-UHFFFAOYSA-N N-benzyloxycarbonylglycine Chemical compound OC(=O)CNC(=O)OCC1=CC=CC=C1 CJUMAFVKTCBCJK-UHFFFAOYSA-N 0.000 description 2
- 108010046645 N-carbobenzoxyglycylproline Proteins 0.000 description 2
- 206010067152 Oral herpes Diseases 0.000 description 2
- 206010053159 Organ failure Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 description 2
- AUQGUYPHJSMAKI-CYDGBPFRSA-N Pro-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 AUQGUYPHJSMAKI-CYDGBPFRSA-N 0.000 description 2
- XZKQVQKUZMAADP-IMJSIDKUSA-N Ser-Ser Chemical group OC[C@H](N)C(=O)N[C@@H](CO)C(O)=O XZKQVQKUZMAADP-IMJSIDKUSA-N 0.000 description 2
- GNVMUORYQLCPJZ-UHFFFAOYSA-M Thiocarbamate Chemical compound NC([S-])=O GNVMUORYQLCPJZ-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 108700010756 Viral Polyproteins Proteins 0.000 description 2
- 108700022715 Viral Proteases Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 150000001263 acyl chlorides Chemical class 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 229960003767 alanine Drugs 0.000 description 2
- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 2
- 125000004414 alkyl thio group Chemical group 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 238000003782 apoptosis assay Methods 0.000 description 2
- 125000005098 aryl alkoxy carbonyl group Chemical group 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 230000037444 atrophy Effects 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- NHYXMAKLBXBVEO-UHFFFAOYSA-N bromomethyl acetate Chemical compound CC(=O)OCBr NHYXMAKLBXBVEO-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000011304 dilated cardiomyopathy 1A Diseases 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 210000003890 endocrine cell Anatomy 0.000 description 2
- 125000003754 ethoxycarbonyl group Chemical group C(=O)(OCC)* 0.000 description 2
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000003818 flash chromatography Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 238000007421 fluorometric assay Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 125000005935 hexyloxycarbonyl group Chemical group 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 description 2
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 2
- XMJHPCRAQCTCFT-UHFFFAOYSA-N methyl chloroformate Chemical compound COC(Cl)=O XMJHPCRAQCTCFT-UHFFFAOYSA-N 0.000 description 2
- 125000004170 methylsulfonyl group Chemical group [H]C([H])([H])S(*)(=O)=O 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- TYRGLVWXHJRKMT-QMMMGPOBSA-N n-benzyloxycarbonyl-l-serine-betalactone Chemical compound OC(=O)[C@H](C)NC(=O)OCC1=CC=CC=C1 TYRGLVWXHJRKMT-QMMMGPOBSA-N 0.000 description 2
- OBPRZLTWBJYCTI-UHFFFAOYSA-N n-hexyl-n-methylcarbamoyl chloride Chemical compound CCCCCCN(C)C(Cl)=O OBPRZLTWBJYCTI-UHFFFAOYSA-N 0.000 description 2
- VFXVAXFIFHSGNR-UHFFFAOYSA-N octyl carbonochloridate Chemical compound CCCCCCCCOC(Cl)=O VFXVAXFIFHSGNR-UHFFFAOYSA-N 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000000816 peptidomimetic Substances 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000005522 programmed cell death Effects 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000023504 respiratory system disease Diseases 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- QCMHWZUFWLOOGI-UHFFFAOYSA-N s-ethyl chloromethanethioate Chemical compound CCSC(Cl)=O QCMHWZUFWLOOGI-UHFFFAOYSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- 150000003456 sulfonamides Chemical class 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 230000007502 viral entry Effects 0.000 description 2
- KUXLVFFUSZCVHJ-YWDSYVAPSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-acetamido-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-methylbutanoyl]amino]butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(C)=O KUXLVFFUSZCVHJ-YWDSYVAPSA-N 0.000 description 1
- XHGIWCXAACQYGK-OALUTQOASA-N (2s)-5-(diaminomethylideneazaniumyl)-2-[[(2s)-3-phenyl-2-(phenylmethoxycarbonylamino)propanoyl]amino]pentanoate Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(=N)N)C(O)=O)NC(=O)OCC=1C=CC=CC=1)C1=CC=CC=C1 XHGIWCXAACQYGK-OALUTQOASA-N 0.000 description 1
- ZMZRKOASUWINDA-VEABSNGSSA-N (4s)-4-[[(2s)-2-amino-3-carboxypropanoyl]amino]-5-[[(2s)-1-[[(2s)-1-carboxy-3-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-oxopentanoic acid Chemical compound OC(=O)C[C@@H](C=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O ZMZRKOASUWINDA-VEABSNGSSA-N 0.000 description 1
- KUHSEZKIEJYEHN-UHFFFAOYSA-N 2-amino-3-hydroxypropanoic acid;2-aminopropanoic acid Chemical compound CC(N)C(O)=O.OCC(N)C(O)=O KUHSEZKIEJYEHN-UHFFFAOYSA-N 0.000 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- POAOYUHQDCAZBD-UHFFFAOYSA-N 2-butoxyethanol Chemical compound CCCCOCCO POAOYUHQDCAZBD-UHFFFAOYSA-N 0.000 description 1
- BXEAAHIHFFIMIE-UHFFFAOYSA-N 3-chlorothiophene-2-carboxylic acid Chemical compound OC(=O)C=1SC=CC=1Cl BXEAAHIHFFIMIE-UHFFFAOYSA-N 0.000 description 1
- SJQRQOKXQKVJGJ-UHFFFAOYSA-N 5-(2-aminoethylamino)naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(NCCN)=CC=CC2=C1S(O)(=O)=O SJQRQOKXQKVJGJ-UHFFFAOYSA-N 0.000 description 1
- CXISPYVYMQWFLE-VKHMYHEASA-N Ala-Gly Chemical group C[C@H]([NH3+])C(=O)NCC([O-])=O CXISPYVYMQWFLE-VKHMYHEASA-N 0.000 description 1
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 229940088872 Apoptosis inhibitor Drugs 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- JWCCFNZJIRZUCL-AVGNSLFASA-N Arg-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N JWCCFNZJIRZUCL-AVGNSLFASA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- JEEFEQCRXKPQHC-KKUMJFAQSA-N Asn-Leu-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JEEFEQCRXKPQHC-KKUMJFAQSA-N 0.000 description 1
- JHFNSBBHKSZXKB-VKHMYHEASA-N Asp-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(O)=O JHFNSBBHKSZXKB-VKHMYHEASA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 108091005502 Aspartic proteases Proteins 0.000 description 1
- 102000035101 Aspartic proteases Human genes 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108091007065 BIRCs Proteins 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- QYEZBGLEAGHANC-UHFFFAOYSA-N CCCCCCOC(=O)C=1SC=CC=1Cl Chemical compound CCCCCCOC(=O)C=1SC=CC=1Cl QYEZBGLEAGHANC-UHFFFAOYSA-N 0.000 description 1
- 108010049990 CD13 Antigens Proteins 0.000 description 1
- CKDWPUIZGOQOOM-UHFFFAOYSA-N Carbamyl chloride Chemical compound NC(Cl)=O CKDWPUIZGOQOOM-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100026549 Caspase-10 Human genes 0.000 description 1
- 102100032616 Caspase-2 Human genes 0.000 description 1
- 102100025597 Caspase-4 Human genes 0.000 description 1
- 102100038918 Caspase-6 Human genes 0.000 description 1
- 102000004039 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 108090000258 Cathepsin D Proteins 0.000 description 1
- 102000003908 Cathepsin D Human genes 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- YXQDRIRSAHTJKM-IMJSIDKUSA-N Cys-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YXQDRIRSAHTJKM-IMJSIDKUSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229940124087 DNA topoisomerase II inhibitor Drugs 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- 229940124213 Dipeptidyl peptidase 4 (DPP IV) inhibitor Drugs 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- OPINTGHFESTVAX-BQBZGAKWSA-N Gln-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N OPINTGHFESTVAX-BQBZGAKWSA-N 0.000 description 1
- MGJMFSBEMSNYJL-AVGNSLFASA-N Gln-Asn-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MGJMFSBEMSNYJL-AVGNSLFASA-N 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- JZDHUJAFXGNDSB-WHFBIAKZSA-N Glu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O JZDHUJAFXGNDSB-WHFBIAKZSA-N 0.000 description 1
- QRWPTXLWHHTOCO-DZKIICNBSA-N Glu-Val-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QRWPTXLWHHTOCO-DZKIICNBSA-N 0.000 description 1
- 108010058940 Glutamyl Aminopeptidase Proteins 0.000 description 1
- 102000006485 Glutamyl Aminopeptidase Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- VPZXBVLAVMBEQI-VKHMYHEASA-N Glycyl-alanine Chemical group OC(=O)[C@H](C)NC(=O)CN VPZXBVLAVMBEQI-VKHMYHEASA-N 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 229910004373 HOAc Inorganic materials 0.000 description 1
- 206010019196 Head injury Diseases 0.000 description 1
- 208000010496 Heart Arrest Diseases 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 1
- 101000983518 Homo sapiens Caspase-10 Proteins 0.000 description 1
- 101000867612 Homo sapiens Caspase-2 Proteins 0.000 description 1
- 101000933112 Homo sapiens Caspase-4 Proteins 0.000 description 1
- 101001033249 Homo sapiens Interleukin-1 beta Proteins 0.000 description 1
- 108010016183 Human immunodeficiency virus 1 p16 protease Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 101700084783 ICP35 Proteins 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102000055031 Inhibitor of Apoptosis Proteins Human genes 0.000 description 1
- 102100039065 Interleukin-1 beta Human genes 0.000 description 1
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 1
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- DEFJQIDDEAULHB-IMJSIDKUSA-N L-alanyl-L-alanine Chemical group C[C@H](N)C(=O)N[C@@H](C)C(O)=O DEFJQIDDEAULHB-IMJSIDKUSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FORGMRSGVSYZQR-YFKPBYRVSA-N L-leucinamide Chemical compound CC(C)C[C@H](N)C(N)=O FORGMRSGVSYZQR-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- NFNVDJGXRFEYTK-YUMQZZPRSA-N Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O NFNVDJGXRFEYTK-YUMQZZPRSA-N 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710159527 Maturation protein A Proteins 0.000 description 1
- 101710091157 Maturation protein A2 Proteins 0.000 description 1
- 101710089759 Melanin-concentrating hormone receptor 2 Proteins 0.000 description 1
- 239000012359 Methanesulfonyl chloride Substances 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Chemical group 0.000 description 1
- 101800001020 Non-structural protein 4A Proteins 0.000 description 1
- MSHZHSPISPJWHW-UHFFFAOYSA-N O-(chloroacetylcarbamoyl)fumagillol Chemical compound O1C(CC=C(C)C)C1(C)C1C(OC)C(OC(=O)NC(=O)CCl)CCC21CO2 MSHZHSPISPJWHW-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- KLAONOISLHWJEE-QWRGUYRKSA-N Phe-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KLAONOISLHWJEE-QWRGUYRKSA-N 0.000 description 1
- KOUUGTKGEQZRHV-KKUMJFAQSA-N Phe-Gln-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O KOUUGTKGEQZRHV-KKUMJFAQSA-N 0.000 description 1
- JXWLMUIXUXLIJR-QWRGUYRKSA-N Phe-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 JXWLMUIXUXLIJR-QWRGUYRKSA-N 0.000 description 1
- VADLTGVIOIOKGM-BZSNNMDCSA-N Phe-His-Leu Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CN=CN1 VADLTGVIOIOKGM-BZSNNMDCSA-N 0.000 description 1
- WEQJQNWXCSUVMA-RYUDHWBXSA-N Phe-Pro Chemical compound C([C@H]([NH3+])C(=O)N1[C@@H](CCC1)C([O-])=O)C1=CC=CC=C1 WEQJQNWXCSUVMA-RYUDHWBXSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000024777 Prion disease Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- NCDNCNXCDXHOMX-UHFFFAOYSA-N Ritonavir Natural products C=1C=CC=CC=1CC(NC(=O)OCC=1SC=NC=1)C(O)CC(CC=1C=CC=CC=1)NC(=O)C(C(C)C)NC(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-UHFFFAOYSA-N 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- VIIJCAQMJBHSJH-FXQIFTODSA-N Ser-Met-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O VIIJCAQMJBHSJH-FXQIFTODSA-N 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 206010043189 Telangiectasia Diseases 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 239000000317 Topoisomerase II Inhibitor Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- DFQZDQPLWBSFEJ-LSJOCFKGSA-N Val-Val-Asn Chemical group CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N DFQZDQPLWBSFEJ-LSJOCFKGSA-N 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- MIFGOLAMNLSLGH-QOKNQOGYSA-N Z-Val-Ala-Asp(OMe)-CH2F Chemical compound COC(=O)C[C@@H](C(=O)CF)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)OCC1=CC=CC=C1 MIFGOLAMNLSLGH-QOKNQOGYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 1
- 239000012346 acetyl chloride Substances 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 230000001270 agonistic effect Effects 0.000 description 1
- 108010047495 alanylglycine Chemical group 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940124411 anti-hiv antiviral agent Drugs 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000000158 apoptosis inhibitor Substances 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000000010 aprotic solvent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 108010051758 aspartyl-glutamyl-valyl-aspartal Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000003778 catagen phase Effects 0.000 description 1
- 101150055276 ced-3 gene Proteins 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- FZFAMSAMCHXGEF-UHFFFAOYSA-N chloro formate Chemical compound ClOC=O FZFAMSAMCHXGEF-UHFFFAOYSA-N 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 230000010405 clearance mechanism Effects 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- IPIVAXLHTVNRBS-UHFFFAOYSA-N decanoyl chloride Chemical compound CCCCCCCCCC(Cl)=O IPIVAXLHTVNRBS-UHFFFAOYSA-N 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- 239000003603 dipeptidyl peptidase IV inhibitor Substances 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000007878 drug screening assay Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 230000008519 endogenous mechanism Effects 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 231100000584 environmental toxicity Toxicity 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 210000004920 epithelial cell of skin Anatomy 0.000 description 1
- 230000010437 erythropoiesis Effects 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 230000003492 excitotoxic effect Effects 0.000 description 1
- 231100000063 excitotoxicity Toxicity 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 125000003983 fluorenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3CC12)* 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 108700004026 gag Genes Proteins 0.000 description 1
- 108010045774 gag protein (129-135) Proteins 0.000 description 1
- 108010027225 gag-pol Fusion Proteins Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 125000000267 glycino group Chemical group [H]N([*])C([H])([H])C(=O)O[H] 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Chemical group OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical group [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- YWGHUJQYGPDNKT-UHFFFAOYSA-N hexanoyl chloride Chemical compound CCCCCC(Cl)=O YWGHUJQYGPDNKT-UHFFFAOYSA-N 0.000 description 1
- KIWBRXCOTCXSSZ-UHFFFAOYSA-N hexyl carbonochloridate Chemical compound CCCCCCOC(Cl)=O KIWBRXCOTCXSSZ-UHFFFAOYSA-N 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000012188 high-throughput screening assay Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000937 inactivator Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011246 intracellular protein detection Methods 0.000 description 1
- 230000004410 intraocular pressure Effects 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- OFCCYDUUBNUJIB-UHFFFAOYSA-N n,n-diethylcarbamoyl chloride Chemical compound CCN(CC)C(Cl)=O OFCCYDUUBNUJIB-UHFFFAOYSA-N 0.000 description 1
- XJINZNWPEQMMBV-UHFFFAOYSA-N n-methylhexan-1-amine Chemical compound CCCCCCNC XJINZNWPEQMMBV-UHFFFAOYSA-N 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 230000010046 negative regulation of endothelial cell proliferation Effects 0.000 description 1
- QAGYKUNXZHXKMR-HKWSIXNMSA-N nelfinavir Chemical compound CC1=C(O)C=CC=C1C(=O)N[C@H]([C@H](O)CN1[C@@H](C[C@@H]2CCCC[C@@H]2C1)C(=O)NC(C)(C)C)CSC1=CC=CC=C1 QAGYKUNXZHXKMR-HKWSIXNMSA-N 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- WIVNTNLDTMNDNO-UHFFFAOYSA-N octane-1-sulfonyl chloride Chemical compound CCCCCCCCS(Cl)(=O)=O WIVNTNLDTMNDNO-UHFFFAOYSA-N 0.000 description 1
- REEZZSHJLXOIHL-UHFFFAOYSA-N octanoyl chloride Chemical compound CCCCCCCC(Cl)=O REEZZSHJLXOIHL-UHFFFAOYSA-N 0.000 description 1
- QGEROENGKYUMGM-UHFFFAOYSA-N octyl 3-chlorothiophene-2-carboxylate Chemical compound CCCCCCCCOC(=O)C=1SC=CC=1Cl QGEROENGKYUMGM-UHFFFAOYSA-N 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000002974 pharmacogenomic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 230000009894 physiological stress Effects 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 208000030761 polycystic kidney disease Diseases 0.000 description 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000003658 preventing hair loss Effects 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000003614 protease activity assay Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000002461 renin inhibitor Substances 0.000 description 1
- 229940086526 renin-inhibitors Drugs 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 210000001116 retinal neuron Anatomy 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- NCDNCNXCDXHOMX-XGKFQTDJSA-N ritonavir Chemical compound N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-XGKFQTDJSA-N 0.000 description 1
- 229960000311 ritonavir Drugs 0.000 description 1
- YPSUCTSXOROPBS-UHFFFAOYSA-N s-methyl chloromethanethioate Chemical compound CSC(Cl)=O YPSUCTSXOROPBS-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000002805 secondary assay Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- XZPVPNZTYPUODG-UHFFFAOYSA-M sodium;chloride;dihydrate Chemical compound O.O.[Na+].[Cl-] XZPVPNZTYPUODG-UHFFFAOYSA-M 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 208000009056 telangiectasis Diseases 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- JLGLQAWTXXGVEM-UHFFFAOYSA-N triethylene glycol monomethyl ether Chemical compound COCCOCCOCCO JLGLQAWTXXGVEM-UHFFFAOYSA-N 0.000 description 1
- GRGCWBWNLSTIEN-UHFFFAOYSA-N trifluoromethanesulfonyl chloride Chemical compound FC(F)(F)S(Cl)(=O)=O GRGCWBWNLSTIEN-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000012036 ultra high throughput screening Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229940023080 viracept Drugs 0.000 description 1
- 230000008478 viral entry into host cell Effects 0.000 description 1
- 230000029302 virus maturation Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06052—Val-amino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1488—Methods for deciding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- This invention is in the field of intracellular detection of enzymes using fluorogenic or fluorescent probes.
- the invention relates to novel fluorescent dyes and application of these dyes for the preparation of novel fluorogenic or fluorescent peptide or amino acid derivatives which are substrates of proteases and peptidases.
- the invention relates to novel fluorogenic or fluorescent peptide derivatives which are substrates of enzymes involved in apoptosis, such as caspases and the lymphocyte-derived serine protease Granzyme
- the invention also relates to a process for measuring the activity of caspases and other enzymes involved in apoptosis in living or dead whole cells, cell lines or tissue samples derived from any healthy, diseased, infected or cancerous organ or tissue.
- the invention also relates to the use of the fluorogenic or fluorescent substrates in a novel assay system for discovering or detecting inhibitors or inducers of apoptosis in compound collections or compound libraries.
- the invention relates to the use of the fluorogenic or fluorescent substrates in determining the sensitivity of cancer cells to treatment with chemotherapeutic drugs.
- the invention also relates to novel fluorogenic or fluorescent peptide derivatives which are substrates of exopeptidases such as aminopeptidase A and N, methionine aminopeptidase and dipeptidyl-peptidase IV, endopetidases such as calpain, proteases such as HIV proteases.
- exopeptidases such as aminopeptidase A and N, methionine aminopeptidase and dipeptidyl-peptidase IV, endopetidases such as calpain
- proteases such as HIV proteases.
- HCMV protease HSV protease
- HCV protease and adenovirus protease.
- Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 2(5:59-86 (1951); Glucksmann, A., Archives de Biologie 76:419-431 (1965); Ellis et al., Dev. 772:591-603 (1991); Vaux et al., Cell 76:111-119 (1994)).
- Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function.
- apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO96/20721).
- Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and
- a cell activates its internally encoded suicide program as a result of either internal or external signals.
- the suicide program is executed through the activation of a carefully regulated genetic program (Wylie et al, Int. Rev. Cyt. 68:251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7:663 (1991)).
- Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529- 536 (1995)).
- IL-1B Mammalian interleukin-l ⁇
- IL-1B plays an important role in various pathologic processes, including chronic and acute inflammation and autoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7, 45-56 (1986)).
- IL-l ⁇ is synthesized as a cell associated precursor polypeptide (pro-IL-l ⁇ ) that is unable to bind IL-1 receptors and is biologically inactive (Mosley et al, J. Biol Chem.
- IL-1 is also a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias (Dinarello. C.A.. Blood 77: 1627-1652 (1991 ); diGiovine et al,
- Interleukin-l ⁇ converting enzyme is a protease responsible for the activation of interleukin-l ⁇ (IL-l ⁇ ) (Thornberry, N.A., et al, Nature 356:16 (1992); Yuan, J., et al. Cell 75:641 (1993)).
- ICE is a substrate-specific cysteine protease that cleaves the inactive prointerleukin-1 to produce the mature IL-1.
- the genes that encode for ICE and CPP32 are members of the mammalian ICE/Ced-3 family of genes which presently includes at least twelve members: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2, MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICErellll.
- This gene family has recently been named caspases (Alnernri, E. S. et.
- a death trigger such as Tumor Necrosis Factor, FAS-ligand, oxygen or nutrient deprivation, viruses, toxins, anti-cancer drugs etc.
- caspases upstream in the cascade e.g. FLICE/MACH/MCH5
- capsases further downstream in the cascade e.g. CPP- 32/Yama/Apopain. Activation of the caspase cascade leads to cell death.
- Caspases are also thought to be crucial in the development and treatment of cancer. There is mounting evidence that cancer cells, while containing caspases. lack parts of the molecular machinery that activate the caspase cascade
- chemotherapeutic drugs can trigger cancer cells to undergo suicide by re-activating the dormant caspase cascade.
- Chemotherapeutic drugs may differ in their capacity to activate the caspase system in different classes of cancers. Moreover, it is likely that anti-cancer drugs differ in their ability to activate the caspase cascade in a given cancer (e.g. lung cancer) and in different patients. In other words, there are differences from one patient to another in the chemosensitivity of, e.g. lung cancer cells, to various anti-cancer drugs.
- the excessive activation of the caspase cascade plays a crucial role in a wide variety of degenerative organ diseases, while a non-functioning caspase system is a hallmark of cancer cells.
- New drugs that inhibit or stimulate the caspase cascade are likely to revolutionize the treatment of numerous human diseases ranging from infectious, cardiovascular, endocrine, kidney, liver and brain diseases to diseases of the immune system and to cancer.
- HTCA high-throughput caspase activation
- HTCA assays should be versatile enough to measure the caspase cascade activity inside any living or whole cell, no matter what its origin might be: Cancer cells, tumor cells, immune cells, brain cells, cells of the endocrine system, cells or cell lines from different organ systems, biopsy samples etc. Furthermore, such HTCA assays should be able to measure—within living or whole cells—the activation or inhibition of any of the caspase enzymes or any other enzymes that are involved in the caspase cascade. Developing such versatile HTCA assays represents a substantial advance in the field of drug screening.
- a potentially important application of a HTCA assay system for measuring intracellular caspase enzymes or any other enzymes involved in apoptosis is chemosensitivity testing of human cancers. It is known that there is a genetic difference in the susceptibility of human cancers to the currently marketed anti-cancer drugs: For example, lung cancer cells in one patient might be sensitive to Drug A, while another patient's lung cancer might be insensitive to Drug A, but sensitive to Drug B. This pharmacogenetic difference in chemosensitivity of cancer cells from different individuals is a well-known phenomenon.
- chemosensitivity testing has not found wide-spread use, because the procedures involved have some inherent technical difficulties: The testing is very time consuming (six or more days per screen) and it requires culturing of the cells prior to screening. The cell culture leads to clonal selection of cells and the cultured cells are then no longer representative of the cancer in the patient.
- a HTCA assay system for quickly measuring intracellular caspase activity could be used to determine very rapidly the chemosensitivity profile of freshly excised cancer cells.
- the assay has a high throughput, it would be feasible to test chemosensitivity of multiple samples taken from the same patient, e.g. from different metastases. This information could then be used to design a treatment regimen using combinations of marketed anti-cancer drugs to which the cells showed greatest sensitivity.
- HTCA assays and reagents for such assays that can be employed in drug discovery or diagnostic procedures to quickly detect and measure the activity of compounds that activate or inhibit the caspase cascade or other enzymes involved in apoptosis in the interior of living or dead whole cells.
- a reagent for this type of cell assay ideally should meet the following conditions: a) there should be a big difference in fluorescence signal between peptide-reporter molecule and reporter molecule after the amide bond in peptide-reporter is cleaved by the caspases or other enzymes involved in apoptosis, preferably the peptide-reporter molecule should be non-fluorescent and most preferably the peptide-reporter molecule should be non-fluorescent and colorless; b) the peptide-reporter molecule should be cell permeable, therefore there should be minimum numbers of hydrophilic groups in the molecule and the size of the molecule should preferably be small; c) the peptide-reporter molecule should preferably not diffuse out of the cell once it permeates the cell membrane; d) the reporter molecule should preferably not diffuse out of the cell once it is liberated from the peptide.
- the method of screening apoptosis inhibitors or inducers in whole cells vs cell-free enzyme assay can also be used for the screening of inhibitors of enzymes other than caspases.
- enzyme inhibitors were first identified by cell- free enzyme assays. Cell cultures were then used for secondary assay to assess activity of the active compounds in intact cells.
- a cell permeable fluorogenic or fluorescent substrate will enable the screening of inhibitors of proteases and peptidases and other enzymes directly in living whole cells.
- AGM-1470 (also known as TNP-470) is an angiogenesis inhibitor in clinical trials for a variety of cancers.
- the mechanism of action of AGM-1470 was recently discovered by two independent research groups (Sin, N., et al. Proc. Natl Acad. Sci. U.S.A. 94:6099-6103 (1997); Griffith, E.C., et al, Chem. Biol. 4:461-471 (1997)). They found that AGM-1470 and analogs are inhibitors of methionine animopeptidase type 2 (MetAP-2). The potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined for a series of AGM-1470 analogs and a significant correlation between the two activities was found.
- angiogenesis inhibitors are known to be able to selectively kill cancer cells
- a cellular screening assay for inhibitors of MetAP-2 may result in novel anti-cancer drugs. Therefore cell permeable fluorogenic or fluorescent substrates for MetAP-2 can be used for the screening of inhibitors of MetAP-2 in endothelial cells which could lead to novel anticancer agents.
- HIV protease inhibitors such as ritonavir and viracept have been shown to be very effective in the treatment of patients infected with HIV. These inhibitors were designed based on the structure of the HIV protease substrate.
- a cell permeable fluorogenic or fluorescent substrate for HIV protease can be used for the screening of HIV protease inhibitors in HIV infected cells which could speed up the process for the discovery of novel HIV protease inhibitors and lead to new and better treatment for HIV infection. Since HIV protease processes viral precursor proteins at a late stage in viral replication, a cell permeable fluorogenic or fluorescent substrate for HIV protease also can be used to screen compounds which inhibit gene transcription or translation, viral entry, or other key proteins in the early stage of HIV infection. The fluorogenic or fluorescent substrates also could be used for diagnosis of HIV infection, which might be more sensitive than the currently available methods.
- cell permeable fluorogenic or fluorescent substrates for cathepsin B can be used for the screening of cathepsin B inhibitors.
- Cell permeable fluorogenic or fluorescent substrates for dipeptidyl-peptidase IV can be used for the screening of dipeptidyl-peptidase IV inhibitors.
- Cell permeable fluorogenic or fluorescent substrates for renin can be used for the screening of renin inhibitors and cell permeable fluorogenic or fluorescent substrates for adenovirus protease or other viral proteases can be used for the screening of adenovirus protease or other viral protease inhibitors.
- U.S. Patent Nos. 4,557,862 and 4,640,893 disclose Rhodamine 1 10 derivatives as fluorogenic substrates for proteinases of the formula:
- R, and R 2 which are the same or different, are selected from the group consisting of amino acids, amino acid derivatives, blocked amino acids, blocked amino acid derivatives, and peptides.
- Exemplary (AA) 2 -Rhodamines and (peptide) 2 -Rhodamines are (Z-Arg) 2 -Rhodamine 1 10, (Arg) 2 -Rhodamine 1 10, (Z- Ala-Arg) 2 -Rhodamine 110, (Z-GLV-Arg) 2 -Rhodamine 110, (Z-Glu-Arg) 2 - Rhodamine 1 10, (Z-Gly-Arg) 2 -Rhodamine 1 10, (Z-Leu-Arg) 2 -Rhodamine 110, (Z-Met-Arg) 2 -Rhodamine 110, (Z-Phe-Arg) 2 -Rhodamine 1 10.
- WO 96/36729 discloses compounds or their salts for assaying the activity of an enzyme inside a metabolically active whole cell.
- the assay compound is said to include a leaving group and an indicator group.
- the leaving group is selected from the group comprising amino acids, peptides, saccharides. sulfates, phosphates, esters, phosphate esters, nucleotides, polynucleotides, nucleic acids, pyrimidines, purines, nucleosides, lipids and mixtures.
- the indicator group is selected from compounds which have a first state when joined to the leaving group, and a second state when the leaving group is cleaved from the indicator group by the enzyme.
- Preferred indicator compounds are said to be Rhodamine 110. rhodol, and fluorescein and analogs of these compounds.
- the patent application listed many enzymes and substrates of enzymes.
- US patent 5,576,424 disclosed haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells of the formula: XR-SPACER-REPORTER-BLOCK
- -BLOCK is a group selected to be removable by action of a specific analyte, to give reporter spectral properties different from those of the substrate
- - REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK- REPORTER bond, has spectral properties different from those of the substrate
- SPACER- is a covalent linkage; and XR- is a haloalkyl moiety that can covalently react with an intracellular thiol to form a thioether conjugate.
- Preferred reporter compounds are said to include Rhodamine- 1 10. rhodol. fluorescein and others.
- the invention relates to fluorogenic or fluorescent reporter compounds of Formula I: x-y-z (I) or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein x and z is the same or different and is a peptide or amino acid or acyl group or other structure such that compounds of Formula I is a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and wherein the scissile bond is only one or both of the x-y and y-z bonds in Formula I when x is the same as z, or wherein the scissile bond is only one of the x-y or y-z bond in Formula I when x is not the same as z.
- y is a fluorogenic or fluorescent moiety.
- Preferred compounds are represented by Formula II:
- R is an N- terminal protecting group such as t-butyloxycarbonyl, acyl, and benzyloxycarbonyl; each AA independently is a residue of any natural or non- natural ⁇ -amino acid or ⁇ -amino acid, or derivatives of an ⁇ -amino acid or ⁇ - amino acid; each n is independently 0-5; and y is a fluorogenic or fluorescent moiety.
- Preferred y is a Rhodamine including Rhodamine 110, Rhodamine 116 and Rhodamine 19. Most preferred y is Rhodamine 110.
- R is t-butyloxycarbonyl, acyl and benzyloxycarbonyl.
- Preferred values of n are 1-3.
- the invention also relates to a method for the preparation of a compound of Formula III, comprising
- -(AA) n is WEH SEQ ID ⁇ O:l, YVA SEQ ID NO:2, LEH SEQ ID NO:3, DET SEQ ID NO:4, DEV SEQ ID NO:5, DEH SEQ ID NO:6, VEH SEQ ID NO:7, LET SEQ ID NO:8, LEV SEQ ID NO:9, SHV SEQ ID NO: 10, DEL SEQ ID NO:l l, DGP SEQ ID NO: 12, DEP SEQ ID NO:13, DGT SEQ ID NO:14, DLN SEQ ID NO:15, DEE SEQ ID NO:16, DSL SEQ ID NO: 17, DVP SEQ ID NO: 18, DEA SEQ ID NO: 19, DSY SEQ ID NO:20, ELP SEQ ID NO:21, VED SEQ ID NO:22, IEP SEQ ID NO:23 or IET SEQ ID NO:24, and the carboxy containing amino acids are protected with an OBu-t group which is removed in the final step.
- Another group of preferred compounds falling within the scope of Formula I include compounds wherein x is not the same as z.
- Preferred compounds of this group include those wherein x is a peptide or other structure which makes the compound a substrate for caspases, or a substrate for other proteases or peptidases or other enzymes; and the x-y bond in Formula I is the scissile bond under biological conditions, z is a blocking group and the y-z bond in Formula I is not a scissile bond under biological conditions.
- novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V:
- R,, AA, n and y are as defined previously in formula II;
- R 6 is a blocking group which is not an amino acid or a derivative of an amino acid.
- R 2 and R are the same or different and are independently hydrogen, alkyl or aryl
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- R,, R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
- R 2 and R are the same or different and are independently hydrogen, alkyl or aryl
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- R ] , R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
- R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl; and R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- the invention also relates to a method for the preparation of a compound of Formula VII, comprising
- the invention also relates to the novel fluorescent dyes of Formula VI which are derivatives of Rhodamines.
- These compounds are prepared by introducing a blocking group R ⁇ into one of the two amino groups of Rhodamine.
- the R 2 HN group in Formula VI provides the point of attachment for reaction with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form a peptide amide bond.
- the reaction will convert the fluorescent molecule of Formula VI into a non-fluorescent peptide-reporter molecule of Formulae VII- IX which is a substrate for a protease or peptidase. Cleavage of the scissile peptide-reporter amide bond in peptide-reporter by proteases or peptidases produces compound of Formula VI or VI' which is fluorescent.
- novel fluorescent dyes of this invention are of Formula VI:
- R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
- R is a blocking group which is not an amino acid or a derivative of an amino acid
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- Preferred R 2 and R 3 are hydrogen, methyl or ethyl; Preferred R 4 and R 5 are hydrogen or methyl.
- the invention also relates to a process of using the reporter compounds represented by Formula I to measure the activity of intracellular caspases or other enzymes involved in apoptosis in living or dead whole cells or tissues.
- the invention also relates to methods of using the compounds represented by Formula I and the assay processes described herein to measure the activation or inhibition of any of the caspase enzymes inside any living or dead whole cell or tissue (normal or cancerous) by a test substance or substances.
- the compounds represented by Formula I are cell-permeable, that is, they can be introduced into whole cells or tissue samples.
- the compounds are fluorogenic or fluorescent and can be designed to be specific for any of the known caspases or for any other intracellular enzymes involved in apoptosis.
- fluorogenic or fluorescent substrates for specific caspases can be synthesized by the procedures described herein.
- the fluorogenic or fluorescent substrates can also be designed to measure more than one enzyme at a time, by designing substrates that are recognized and cleaved by more than one of the enzymes involved in the caspase cascade. Fluorogenic or fluorescent substrates which are "promiscuous" for more than one caspase may be utilized using the assay process described herein to measure the activity of as yet unknown caspases.
- the fluorogenic or fluorescent reporter molecules described herein are cleaved and respond with a large increase in fluorescence emission.
- the change in fluorescence can be measured spectrofluorometrivally.
- the reporter molecules can also be used to measure baseline caspase activity in cells that are not undergoing apoptosis.
- the method is easily adaptable to high throughput or ultra- high throughput screening assays.
- the assay system is very versatile. Examples of the extreme versatility of the assay system are given below:
- the assay can be used to screen a cell or tissue for baseline activity of any caspase enzyme or any other enzyme involved in apoptosis. 2.
- the assay can be used with equal ease to screen for compounds that can either activate or inhibit the caspase cascade. That means the assay can be used to screen for drugs against degenerative diseases or for drugs against cancer.
- the assay can be used to screen for caspase cascade activation or inhibition in any living or dead cells or cell lines derived from any organ system in the body including, but not limited to. hair, brain, peripheral nervous system, eye, ear. nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands, joints, bones and skin.
- the assay can be used to screen for drugs with potential use in any disease of any organ system in the body that involves malfunction of the caspase cascade.
- the assay can be used to screen for drugs that might modulate the caspase cascade directly or indirectly, i.e. by modulating the caspases itself or by modulating cellular receptors and co-factors that influence the caspase cascade.
- the assay can be used to determine the site of action at which a caspase cascade modulator interferes. That is, the assay can help to pin down the molecular mechanism of action of a novel caspase cascade modulator drug.
- the invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I for finding new compounds or new uses for known compounds in reducing, preventing or treating maladies in which apoptotic cell death is either a causative factor or a result.
- uses for the present invention include screening for compounds that can protect the nervous system following focal ischemia and global ischemia: screening for compounds that can treat neurodegenerative disorders such as Alzheimer's disease, Huntington' s Disease, prion diseases, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia, telangiectasia, and spinobulbar atrophy; screening for compounds that can treat heart disease including myocardial infarction, congestive heart failure and cardiomyopathy; screening for compounds that can treat retinal disorders; screening for compounds that treat autoimmune disorders including lupus erythematosus, rheumatoid arthritis, type I diabetes, Sj ⁇ gren's syndrome and glomerulonephritis; screening for compounds that
- the present invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I in screening procedures where libraries of known drugs or combinatorial or other compound libraries are screened for compounds with anti-tumor or anti-cancer activity.
- the cancer cells or cell lines can be derived from any cancer of any internal or external organ system in the body including, but not limited to brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (e.g. prostate gland), joints, bones and skin.
- brain peripheral nervous system
- eye ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal
- the present invention also relates to the use of the fluorogenic or fluorescent substrates represented by Formula I in diagnostic procedures to determine the chemosensitivity or resistance of cancer cells taken from an animal or a human being to treatment with chemotherapeutic drugs.
- the cancer cells or cell lines can be derived from any cancer of any internal or external organ system in the body including, but not limited to brain, peripheral nervous system, eye, ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal tract, pancreas, endocrine glands and tissues, kidney, bladder, reproductive organs and glands (e.g. prostate gland), joints, bones and skin.
- brain peripheral nervous system
- eye ear, nose, mouth, tonsils, teeth, esophagus, lung, heart, blood, blood vessels, bone marrow, lymph nodes, thymus, spleen, immune system, liver, stomach, intestinal
- the invention relates to a method for detecting an enzyme involved in the apoptosis cascade in one or more cells, comprising (a) contacting the one or more cells with a reporter compound according to the invention under conditions whereby the reporter compound is taken into said one or more cells, and
- the invention also relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptosis cascade in one or more test cells, comprising
- test cells may be contacted with said test substance prior to, after, or substantially simultaneously with the reporter compound according to the invention.
- the method may be used to detect whether the test substance stimulates or inhibits the activity of the enzyme.
- the invention also relates to further contacting the test cells with a second test substance or mixture of test substances in the presence of the first test substance.
- the invention also relates to a method to determine the sensitivity of an animal with cancer to treatment with one or more chemotherapeutic agents, comprising
- the invention also relates to a method to monitor the treatment of an animal with one or more chemotherapeutic drugs, comprising (a) administering one or more chemotherapeutic drugs to the animal,
- the animal may suffer from a malady in which apoptotic cell death is either a causative factor or a result.
- the invention also relates to a method for determining whether a test substance inhibits or prevents cell death in one or more test cells, comprising
- test substance either interacts with an external membrane receptor or is taken into the cell and the reporter compound is taken into the cell
- the invention also relates to a method for determining whether a test substance causes or enhances cell death in one or more test cells, comprising
- test substance (a) contacting the test cells with the test substance and the reporter compound according to the invention under conditions whereby the test substance either interacts with an external membrane receptor or is taken into the cells and the reporter compound is taken into the cells,
- the reporter compounds represented by Formula IX are cell-permeable, that is they can be introduced into whole cells.
- the compounds are fluorogenic or fluorescent and can be designed to be specific for the known enzymes of interest, such as methionine aminopeptidase or HIV protease.
- the invention also relates to a method for detecting a viral protease in one or more cells, comprising
- the invention also relates to a method for measuring the activity of a viral protease in one or more viral infected cells, comprising
- the invention also relates to a method for determining whether a test substance has an effect on the activity of viral protease in one or more viral infected cells, comprising
- the cells are HIV infected cells and the viral protease is HIV protease.
- the cells are adenovirus infected cells and the viral protease is adenovirus protease.
- the cells are HSV infected cells and the viral protease is
- the cells are HCMV infected cells and the viral protease is HCMV protease. In another preferred embodiment, the cells are HCV infected cells and the viral protease is HCV protease.
- the invention also relates to a method for measuring the activity of protease or peptidase in cells, comprising (a) contacting the test cells with the reporter compound according to the invention under conditions whereby the reporter compound is taken into the test cells, or the reporter compound is interacting with an external membrane protease or peptidase of said cells, and (b) recording the fluorescence of the cells, wherein a change or increase in fluorescence within the test cell compared to control cells which have not been so contacted is a measure of the activity of the the protease or peptidase.
- the invention also relates to a method for determining whether a test substance has an effect on the activity of protease or peptidase in the test cells, comprising
- the cells are endothelial cells and the peptidase is type 2 methionine aminopeptidase.
- cells are T cells and the peptidase is dipeptidyl peptidase-IV.
- the cells are neuron cells and the protease is calpain.
- Figs. 1A-1F depict photographs of HL-60 cells stained by N- octyloxycarbonyl-Rhodamine 110 (Fig. 1A), N-decyloxycarbonyl-Rhodamine- 1 10 (Fig. IB), N-dodecyloxycarbonyl-Rhodamine-1 10 (Fig. 1C), N- hexyloxycarbonyl-Rhodamine-110 (Fig. ID), N-(ethylthio)carbonyl-Rhodamine 1 10 (Fig. IE) and Rhodamine 110 (Fig. IF).
- Figures 2A-2L depict the bar graphs of cleavage of the caspase substrates N-Z-VD-N'-ethoxycarbonyl-Rl 10, N-Z-EVD-N'-ethoxycarbonyl-Rl 10, N-Z- DEVD-N'-ethoxycarbonyl-Rl 10 SEQ ID ⁇ O:5, N-Ac-DEVD-N'-ethoxycarbonyl-
- R110 SEQ ID ⁇ O:5, N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5, N- Ac-DEVD-N'-hexyloxycarbonyl-Rl lO SEQ ID NO: 5. and N-Z-DEVD-N- (ethylthio)carbonyl-RHO SEQ ID ⁇ O:5, by r-caspase-3 (Figs. 2A, 2B,_ 2D, 2G and 2J) and Vinblastine treated HL-60 cell lysates (Figs. 2C, 2E, 2H and 2K) compared to HL-60 control (DMSO treated) lysates (Figs. 2F, 21 and 2L).
- Figs. 3A-3E depict photographs of cells stained by incubation with N-Ac-
- DMSO (Fig. 3B) treated HL-60 cells, vinblastine treated HL-60 cells with N-Ac-
- Fig. 4 depicts a graph showing the results of a cleavage assay of N-Ac- DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 by antiFas and PBS treated Jurkat cells.
- Fig. 5 depicts a bar graph showing the results of a cleavage assay of N-Ac- LEVD-N'-ethoxycarbonyl-Rl 10 SEQ ID ⁇ O:5 by caspase-3, -6, -7 and -8.
- Fig. 6 depicts a bar graph with the results of a cleavage assay of N-Z-G-N- octyloxycarbonyl-RHO and N-G-N'-octyloxycarbonyl-Rl 10 by HL-60 cell lysates.
- Figures 7A-B depict photographs of HL-60 cells treated with ⁇ -Z-G- ⁇ '- octyloxycarbonyl-Rl 10 (A) and ⁇ -G- ⁇ '-octyloxycarbonyl-Rl 10 (B).
- the fluorogenic or fluorescent substrates of the present invention are compounds having the general Formula I: x-y-z (I) or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein x and z is the same or different and is a peptide or amino acid or acyl group or other structure such that Formula I is a substrate for caspases, or other proteases or peptidases or other enzymes; and wherein the scissile bond is only one or both of the x-y and y- z bonds in Formula I when x is the same as z, or wherein the scissile bond is only one of the x-y or y-z bonds in Formula I when x is not the same as z.
- y is a fluorogenic or fluorescent moiety.
- Preferred compounds falling within the scope of Formula I include compounds wherein x is the same as z. and the first amino acid attached to y is an
- x is the same as z and is a N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O: l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3.
- DGTD SEQ ID NO: 14 DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO:24 or a N-blocked tetrapeptide substrate of granzyme B including IEPD SEQ ID NO: 23 and VEPD SEQ ID NO:27; or x is the same as z and is a N-blocked peptide which corresponds to a carboxyterminal or aminoterminal fragment consisting of 1 , 2 or 3 amino acids of the tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O:l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3, DETD SEQ ID NO:4, DEVD SEQ ID NO:5, DEHD SEQ ID NO:6, VEHD SEQ
- DGPD SEQ ID NO: 12 DEPD SEQ ID NO: 13, DGTD SEQ ID NO: 14, DLND SEQ ID NO:15, DEED SEQ ID NO:16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO:24 and granzyme B including IEPD SEQ ID NO:23 and VEPD SEQ ID NO:27.
- Preferred compounds falling within the scope of Formula I include compounds wherein y is Rhodamine 110.
- pro-reporter molecules such as methyl ester form of carboxyl-containing amino acid residues
- R is an ⁇ - terminal protecting group including t-butyloxycarbonyl. acetyl. benzyloxycarbonyl
- each AA independently is a residue of any natural or non- natural ⁇ -amino acid or ⁇ -amino acid, or derivatives of an ⁇ -amino acid or ⁇ - amino acid: each n independently is 0-5; and y is a fluorogenic or fluorescent moiety.
- An example of a pro-reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising compounds of Formula II.
- AM esters of carboxyl-containing compounds are known to be cell permeable and can be hydrolyzed by esterases inside the cells. Once hydrolyzed, the carboxyl- containing compounds become cell impermeable and are trapped inside the cells
- AM esters can be prepared by reacting the corresponding carboxy-containing compounds with bromomethyl acetate.
- R, AA, n are as defined previously in Formula II.
- R is t-butyloxycarbonyl, acetyl and benzyloxycarbonyl. Also preferred values for n are 1 -3.
- Another group of preferred compounds falling within the scope of Formula I include compounds wherein x is not the same as z.
- Preferred compounds of this group include those wherein x is a peptide or other structure which makes the compound a substrate for caspases, or other proteases or peptidases or other enzymes; and the x-y bond in Formula I is the scissile bond under biological conditions; z is a blocking group and the y-z bond in Formula I is not a scissile bond under biological conditions.
- x is a N-blocked tetrapeptide substrate of a caspase including WEHD SEQ ID ⁇ O: l, YVAD SEQ ID NO:2, LEHD SEQ ID NO:3, DETD SEQ ID NO:4, DEVD SEQ ID NO:5, DEHD SEQ ID NO:6, VEHD SEQ ID NOJ, LETD SEQ ID NO:8, LEHD SEQ ID NO:3, SHVD SEQ ID NO: 10, DELD SEQ ID NO: l 1 , DGPD SEQ ID NO:12.
- DEPD SEQ ID NO: 13 DGTD SEQ ID NO: 14, DLND SEQ ID NO: 15, DEED SEQ ID NO: 16, DSLD SEQ ID NO: 17, DVPD SEQ ID NO: 18, DEAD SEQ ID NO: 19, DSYD SEQ ID NO:20, ELPD SEQ ID NO:21, VEID SEQ ID NO:26, IETD SEQ ID NO: 24 or a N-blocked tetrapeptide substrate of granzyme B including IEPD SEQ ID ⁇ O:23 and VEPD SEQ ID NO:27; or x is a N-blocked peptide which corresponds to a carboxyterminal or aminoterminal fragment consisting of 1.
- novel fluorogenic or fluorescent reporter compounds of this invention are of Formula V:
- R is an N-terminal protecting group including t-butyloxycarbonyl, acetyl, octanoyl and benzyloxycarbonyl; each AA independently is a residue of any natural or non-natural ⁇ -amino acid or ⁇ -amino acid, or a derivative of an ⁇ -amino acid or ⁇ -amino acid; n is 0-5; y is a fluorogenic or fluorescent moiety; and R ⁇ is a blocking group which is not an amino acid or a derivative of an amino acid.
- novel fluorogenic or fluorescent reporter molecules of this invention of Formula VII-IX are derivatives of Rhodamines including Rhodamine 110, Rhodamine 116 and Rhodamine 19.
- These novel fluorogenic or fluorescent reporter molecules are prepared by first introducing a blocking group R,; into one of the two amino groups of a Rhodamine to give novel fluorescent dyes of the Formula VI.
- the remaining HNR 2 group is used for reaction with a potential enzyme substrate to give a fluorogenic substrate of Formula VII-IX.
- By blocking one of the two amino groups in a Rhodamine the overall size of the substrate is reduced compared to a bis-substituted Rhodamine, such as a bis- peptide-Rhodamine.
- the blocking group is selected such that a) it is stable and will not hydrolyze under biological conditions, thus amino acids are excluded because the peptide bond formed can potentially be cleaved by peptidases which are present in the cells; b) it is preferably not too bulky (e.g. is small) in order to reduce the overall size of the peptide-reporter molecule so that it will be a better enzyme substrate; c) it is preferrably hydrophobic in nature so as to increase the cellular permeability of the fluorogenic or fluorescent reporter molecule.
- Preferred R 6 blocking groups include, but are not limited to. an C 2 ..
- alkyloxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a C 2.12 (alkylthio)carbonyl group such as (ethylthio)carbonyl, (hexylthio)carbonyl, (octylthio)carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl, a C 2 .
- acyl (alkanoyl) group such as acetyl and octanoyl, a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl. and an alkyl, haloalkyl or aralkyl sulfonyl group such as methanesulfonyl.
- R 6 blocking groups are the ones that contain a hydrophobic group similar to membrane lipid, thus increasing the cellular permeability of the fluorogenic or fluorescent reporter molecules, as well as retention of the fluorescent moiety in the cells after the cleavage of substrate by targeted protease or peptidase.
- novel fluorogenic or fluorescent reporter molecules of Formula VII- IX are prepared by reacting the amino group NHR 2 of the novel fluorescent dyes of Formula VI with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form an peptide amide bond.
- the reaction converts the fluorescent molecule of Formula VI into a non-fluorescent peptide-repoter molecule of Formulae VII-IX which is a substrate for a protease or peptidase. It is therefore very important that the blocking group R 6 - ⁇ bond of Formula VII should not be cleaved and that the peptide-reporter amide bond should be the scissile bond under biological conditions. Cleavage of the scissile peptide- reporter amide bond of Formulae VII-IX by proteases or peptidases produces a compound of Formula VI or VI' which is fluorescent.
- Formula VII or biologically acceptable salts or pro-reporter molecules (such as methyl ester form of carboxyl-containing amino acid residues) thereof, wherein: R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl; R 6 is a blocking group which is not an amino acid or a derivative of an amino acid;
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- R is an N-terminal protecting group; each AA independently is a residue of any natural or non-natural ⁇ -amino acid or ⁇ -amino acid, or a derivative of an ⁇ -amino acid or ⁇ -amino acid; n is 0-5; and the scissile bond is the Asp- ⁇ bond in Formula VII.
- R 2 and R 3 are hydrogen, methyl or ethyl
- Preferred R 4 and R 5 are hydrogen or methyl.
- Preferred amino acids include the natural amino acids including tyrosine, glycine, phenylalanine, methionine. alanine. serine, isoleucine, leucine, threonine, valine, proline, lysine, histidine, glutamine, glutamic acid, tryptophan, arginine, aspartic acid, asparagine, and cysteine.
- ⁇ on-natural amino acids include t- butylglycine and ⁇ , ⁇ -dimethylglutamine.
- pro-reporter molecule is the methyl ester form of carboxyl-containing amino acid residues comprising compounds of Formula VII.
- Another example of a pro-reporter molecule is the acetoxymethyl (AM) ester form of carboxyl-containing amino acid residues of compounds of Formula VII.
- R,, R 6 , AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
- R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- Compounds of Formula VIII are novel fluorogenic or fluorescent substrates for caspases or other enzymes related with apoptosis.
- cleavage of the amide bond between Asp and Rhodamine will convert the fluorogenic substrate into the fluorescent dye of Formula VI.
- cleavage of the amide bond between Asp and (AA) m will leave the Rhodamine attached to NH 2 -(AA) m .
- the remaining amino acids (AA) may be designed to correspond with the P' sequence of the cleavage site of substrates of caspases or apoptosis related enzymes.
- pro-reporter molecule is the methyl or ethyl ester forms of carboxyl-containing amino acid residues comprising compounds of Formula VIII.
- Another example of a pro-reporter molecule is the acetoxymethyl (AM) or pivaloyloxymethyl (PM) ester form of carboxyl-containing amino acid residues of compounds of Formula VIII.
- AM esters of carboxyl-containing compounds are known to be cell permeable and can be hydrolyzed by esterases inside the cells. Once hydrolyzed, the carboxyl-containing compounds become cell impermeable and are trapped inside the cells (Adams et al, J. Am. Chem. Soc. 117:7957-7968 (1989)).
- AM esters can be prepared by reacting the corresponding carboxy- containing compounds with bromomethyl acetate.
- R] Re, AA and n are as defined previously in Formulae II and V; m is an integer from 0-3.
- R 2 and R 3 are the same or different and are independently hydrogen, alkyl or aryl;
- R 4 and R 5 are the same or different and are independently hydrogen or alkyl.
- Preferred R is t-butyloxycarbonyl, acetyl, octanoyl, dodecanoyl and benzyloxycarbonyl.
- Preferred n is 1-4.
- Preferred R 2 and R 3 are hydrogen, methyl or ethyl.
- Preferred R 4 and R 5 are hydrogen or methyl.
- Preferred R ⁇ blocking groups include, but are not limited to, an C 2 _ 12 alkyloxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl, hexyloxycarbonyl, octyloxycarbonyl, decyloxycarbonyl and dodecyloxycarbonyl; a C 2.12 (alkylthio)carbonyl group such as (ethylthio)carbonyl, (hexylthio)carbonyl, (octylthio)carbonyl; an arylalkyloxycarbonyl group such as benzyloxycarbonyl; a C 2 _ 12 acyl (alkanoyl) group such as acetyl and octanoyl; a carbamyl group such as dimethylcarbamyl, N-methyl-N-hexylcarbamyl; and an alkyl, haloalkyl or aralkyl sulfonyl
- (AA) is designed to be an amino acid or a peptide which is recognized by a specific peptidase or protease as the sequence in the p side and will be cleaved by the targeted peptidase or protease.
- (AA) m is designed to be an amino acid or peptide which is recognized by a specific peptidase or protease as the sequence in the P' side, and which can be removed by aminopeptidases presented in the cells.
- compounds of Formula IX are substrates for endopeptidases such as cathepsin D or protease such as HIV protease: when R, is H, compounds of Formula IX are substrates for exopeptidases such as methionine aminopeptidase.
- compounds of Formula IX are designed to be substrates of type 2 methionine aminopeptidase (MetAP-2). MetAP-2 was identified recently by two research groups (Griffith, E.C., et al, Chem. Biol 4:461-471 (1997) and Sin, ⁇ ., et al, Proc.
- MetAP-2 is a bifunctional enzyme which also regulate protein synthesis by affecting the phosphorylaton state of eIF-2.
- AGM- 1470 is reported to only inhibit the aminopeptidase activity of MetAP-2 and have no effect on the regulatory activity of MetAP-2 (Griffith, E.C., et al, Chem. Biol. 4:461-471 (1997)). Since angiogenesis inhibitor such as AGM-1470 is known to be able to selectively kill cancer cells, inhibitors of MetAP-2 are expected to have anti-angiogenic properties and to be potential novel anticancer agents.
- MetAP-2 is a cobalt-dependent enzyme that hydrolyzes the aminoterminal methionine from certain proteins. Its preferred substrates are Met-X-Y.
- X is an amino acid with small and uncharged side groups, such as Gly, Ala, Ser, whereas Leu, Met, Arg and Tyr are known to result in inactive substrates.
- Y can be Ser, Met, Gly or other amino acids (Li, X. & Chang Y.-H., Biochem. Biophy. Res. Com. 227. 152-159 (1996)). Since Rhodamine is much larger than an amino acid, a compound with methionine directly attached to Rhodamine most probably will not be a substrate for MetAP-2.
- a (AA) m sequence between methionine and Rhodamine will make a good substrate for MetAP-2.
- This type of substrate is expected to work well in a whole cell assay but otherwise will not work in a cell-free MetAP-2 enzyme assay.
- preferred R is H
- preferred (AA) n is Met
- preferred (AA) m is Gly, Ala, Gly- Gly, Ala-Gly or Gly-Ala.
- the methionine will be cleaved by type 2 methionine aminopeptidase in endothelial cells to give the Rhodamine attached to (AA) m . Aminopeptidases present inside the cells will then remove the (AA) m to give the fluorescent dye of Formula VI.
- Compounds of Formula IX will be used for the screening of inhibitors of MetAP-2 in endothelial cells, which is expected to lead to the identification of novel anti-cancer drugs.
- HIV protease is an aspartic protease which processes polypeptides transcribed from the gag and pol genes and is essential for the maturation of infectious virus. Therefore HIV protease has been one of the major targets for chemotherapeutic intervention of HIV.
- HIV protease inhibitors have shown great potential in the treatment of HIV and have been approved for marketing. Most of these HIV protease inhibitors were designed based on the structure of the substrates of the protease. Therefore these compounds are either peptides or peptidomimetics. The search for new and novel HIV protease inhibitors is expected to provide more efficacious drugs for the fight against this deadly disease.
- the preferred substrates of HIV protease are peptides with a scissile hydrophobic-hydrophobic or aromatic-proline peptide bond between the P P)'
- Gln-Arg SEQ ID NO:30 Arg-Gln-Ala-Asn-Phe-Leu-Gly SEQ ID NO:31, Pro- Gly-Asn-Phe-Leu-Gln-Ser SEQ ID NO:32, Ser-Phe-Ser-Phe-Pro-Gln-Ile SEQ ID NO:33, Thr-Leu-Asn-Phe-Pro-Ile-Ser SEQ ID NO:34, Ala-Glu-Thr-Phe-Tyr-Val- Asp SEQ ID NO:35 and Arg-Lys-Val-Leu-Phe-Leu-Asp SEQ ID NO:36.
- HIV protease substrates have been developed, and these include the fluorogenic N-alpha-benzoyl-Arg-Gly-Phe-Pro-MeO-beta-naphthylamide SEQ ID ⁇ O:37, which contains the Phe-Pro dipeptide bond recognized by HIV-1 protease (Tyagi, S.C., and Carter, C.A., Anal. Biochem.
- Glu-Ala, Phe-Glu, or Phe; or preferred (AA) peer is Pro-Phe-His-Leu SEQ ID NO:50, or Phe-His-Leu
- preferred (AA) m is Leu-Glu-Glu-Ser SEQ ID NO:40, Leu- Glu-Glu, Leu-Glu, or Leu
- preferred (AA) ⁇ is Ser-Gln-Asn-Leu-Phe SEQ ID NO: 140, Gln-Asn-Leu-Phe SEQ ID NO:51, Asn-Leu-Phe, Arg-Lys-Ile-Leu-Phe SEQ ID NO: 52, Lys-Ile-Leu-Phe SEQ ID NO: 53, or Ile-Leu-Phe, and preferred
- (AA) m is Leu-Asp-Gly-NH 2 , Leu-Asp-NH 2 . or Leu-NH 2 . More preferred (AA) thread is Ser-Leu-Asn-Phe SEQ ID NO: 54, or Leu-Asn-Phe, and more preferred (AA), situation is Pro-Ile-Val, Pro-He, or Pro; or more preferred (AA) thread is Arg-Gly-Phe, and more preferred (AA) m is Pro. Substrates of HIV protease of Formula IX are expected to work in whole cell assays but otherwise will not work in cell-free enzyme assays.
- HIV protease processes viral precursor proteins at a late stage in viral replication
- a cell permeable fluorogenic or fluorescent substrate for an HIV protease also can be used to screen compounds which inhibit gene transcription or translation, viral entry, or other key proteins in the early stage of HIV infection. Therefore this method can lead to the identification of inhibitors of HIV infections with a novel mechanism, which could not be identified in a cell-free enzyme assay.
- substrates of Formula V also can be used for the diagnosis of HIV infection.
- Compounds of Formula IX also can be designed to be substrates of adenovirus protease.
- Adenovirus are the cause of several diseases including sporatic respiratory disease and epidemic acute respiratory disease which can lead to preumonia.
- Adenovirus protease is a cysteine protease which cleaves several viral proteins and is required for virus maturation and infectivity (Weber, J.M., Curr. Top. Microbiol Immunol. 199/1:221-235 (1995)).
- the preferred substrates of adenovirus protease includes (M.LJ)XGX-G and (M,L,I)XGG-X.
- the specificity of the substrates are mainly determined by P 2 and P 4 amino acids (Diouri, M., et al, J. Biol. Chem. 277:32511-32514 (1996)). Hydrophobic amino acids such as Met, Leu and He are perferred in P 4 . Small amino acid such as Gly is preferred in P 2 . A small and hydrophobic amino acid is also preferred for P, and P,', such as Ala and Gly; while P 3 can accommodate almost any amino acid.
- fluorogenic or fluorescent substrates of adevovirus protease can be designed to incorporate amino acids either from the P side only, or from both the P side and P' side of adenovirus protease substrate for application in whole cell assays.
- preferred R is acetyl or Cbz
- preferred (AA) conflict is Leu-Arg-Gly-Gly SEQ ID NO:55, Met-Arg-Gly-Gly SEQ ID NO:56, Ile-Arg-Gly-Gly SEQ ID NO:57,
- Compounds of Formula IX will be used for the screening of inhibitors of adenovirus protease in adenovirus infected cells.
- Compounds of Formula IX also can be designed to be substrates of herpes simplex virus type 1 (HSV-1) protease.
- HSV-1 protease Human herpes simplex virus type 1 is responsible for herpes labialis (cold sores).
- the HSV-1 protease is a serine protease and is responsible for proteolytic processing of itself and ICP35 for assembly of viral capside (Gao, M, et al, J. Virol 68:3102-3112 (1994)).
- Two proteolytic sites have been identified to be Ala247 and Ser248 and Ala610 and
- preferred R is acetyl or Cbz
- preferred (AA) n is Leu-Val-Leu-Ala SEQ ID NO:62
- Compounds of Formula IX will be used for the screening of inhibitors of HSV-1 protease in HSV-1 infected cells.
- Compounds of Formula IX also can be designed to be substrates of human cytomegalovirus (HCMV) protease.
- HCMV can cause life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients.
- Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein and is essential for virus replication, therefore it is a potential target for therapeutic intervention.
- the HCMV protease is a serine protease and two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). (Sztevens, J.T., et al, Eur. J. Biochem. 226:361-361 (1994)).
- Val-Asn-Ala-Ser-Ser-Arg-Leu-Ala-EDANS SEQ ID NO:63 was synthesized and found to be cleaved efficiently by CMV protease at the Ala-Ser peptide bond (Holskin, B.P., et al, Anal. Biochem. 227:148-155 (1995)). Recently, it was reported that replacement of the Val-Val-Asn sequence corresponding to the P 4 -P 2 residues of the maturation site of the enzyme by the optimized Tbg-Tbg-
- R is acetyl or Cbz
- preferred (AA) n is Val-Val-Asn-Ala SEQ
- HCV hepatitis C virus
- HCV hepatitis C virus
- HCV protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein, thus the NS3/4A protease complex is an attractive target for antiviral therapy against HCV.
- the HCV protease is a serine protease and Cys-Ser has been identified as a cleavage site.
- One of the substrate sequence is Asp-Asp-Ile-Val-Pro-Cys-Ser- Met-Ser-Tyr SEQ ID NO:66, and P, Cys and P 3 Val were found to be critical (Zhang, R., et al, J. Virol. 77:6208-6213 (1997)).
- fluorogenic or fluorescent substrates of HCV protease are designed to incorporate amino acids both from the P side and
- preferred R is acetyl or Cbz
- preferred (AA) is Asp-Asp-Ile-Val-Pro-Cys SEQ ID NO:67, Asp-Ile-Val-Pro-Cys SEQ ID NO:68, or Ile-Val-Pro-Cys SEQ ID NO:69
- preferred (AA) m is Ser-Met-Ser-Tyr SEQ ID NOJ0, Ser-Met-Ser, Ser-
- Formula IX will be used for the screening of inhibitors of HCV protease in HCV infected cells.
- the invention also relates to novel compounds of Formula VI which are derivatives of a Rhodamine and are obtained by introducing a blocking group R 6 onto one of the two amino groups on a Rhodamine.
- the R 2 HN group in Formula VI provides the point of attachment for the reaction with a potential enzyme substrate, such as the carboxylic group of a N-blocked peptide, to form an peptide amide bond.
- the reaction converts the fluorescent molecule of Formula VI into a non-fluorescent molecule of Formulae VII-IX and produces a peptide-reporter molecule which functions as a substrate for a protease or peptidase.
- the peptide- reporter amide bond in Formulae VII-IX is the scissile bond under biological conditions.
- the blocking group can incorporate a hydrophobic group.
- the hydrophobic group is designed to increase the membrane permeability of the substrates, and to result in an accumulation of the substrate inside the cells, as well as to increase retention of the fluorescence moiety inside the cells after its cleavage by targeted protease or peptidase.
- novel fluorescent dyes of this invention are of Formula VI:
- R 2 and R 3 are hydrogen, methyl or ethyl
- Preferred R 4 and R 5 are hydrogen or methyl.
- Compounds of Formula VI of the present invention may exist in tautomeric forms, particularly the ring opening form of Formula VI'.
- the invention includes all tautomeric forms including VI and VI'.
- Preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula II and include, but are not limited to: (Z-WEHD) 2 -Rhodamine 110, SEQ ID NO:l (Z-YVAD) 2 -Rhodamine 110, SEQ ID NO:2 (Z-DETD) 2 -Rhodamine 110, SEQ ID NO:4 (Z-DEVD) 2 -Rhodamine 1 10, SEQ ID NO:5 (Z-DEHD) 2 -Rhodamine 110, SEQ ID NO:6 (Z-VEHD) 2 -Rhodamine 1 10, SEQ ID NOJ (Z-LETD) 2 -Rhodamine 1 10, SEQ ID NO: 8 (Z-LEHD) 2 -Rhodamine 110, SEQ ID NO:3 (Z-LEVD) 2 -Rhodamine 110, SEQ ID NO:9 (Z-IEPD) 2 -Rhodamine 110, SEQ ID NO:23 (Z-VEPD
- Preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula VII and include, but are not limited to:
- N-(Z-YVAD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:2
- SEQ ID ⁇ O:9 N-(Z-DETD)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:4
- SEQ ID NO: 8 N-(Z-IEPD)-.V -acetyl-Rhodamine 110.
- N-(Z-SHVD)-N '-acetyl-Rhodamine 110 SEQ ID NO: 10
- N-(Z-DGPD)-N '-acetyl-Rhodamine 110 SEQ ID NO: 12
- SEQ ID NO: 13 N-(Z-DEPD)-N '-acetyl-Rhodamine 110, SEQ ID NO: 13
- N-(Z-DSLD)-N'-acetyl-Rhodamine 110 SEQ ID NO: 17 N-(Z-DVPD)-N '-acetyl-Rhodamine 110, SEQ ID NO : 18 N-(Z-DEAD)-N '-acetyl-Rhodamine 110, SEQ ID NO: 19 N-(Z-DSYD)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:20 N-(Z-ELPD)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ O:21 N-(Z-VEID)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ O:26 N-(Z-IETD)-N -acetyl-Rhodamine 110, SEQ ID ⁇ O:24
- N-(Z-TD)-N '-acetyl-Rhodamine 110 N-(Z-AD)-N '-acetyl-Rhodamine 110
- N-(Z-VAD)-N '-acetyl-Rhodamine 110 N-(Boc-WEHD)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ O:l
- SEQ ID ⁇ O:2 N-(Ac-LETD)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ O:8 N-(Ac-LEHD)-N '-acetyl-Rhodamine 110.
- SEQ ID ⁇ O:3 N-(Z-DEVD)-N'-methoxycarbonyl-Rhodamine 1
- SEQ ID ⁇ O:2 N-(Ac-LETD)-N '-acetyl-Rhodamine 1 10
- SEQ ID ⁇ O:8 N-(Ac-LEHD)-N '-acetyl-Rhodamine 110.
- SEQ ID ⁇ O:3 N-(
- N-(Z-LEVD)-N'-methoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:9 N-(Z-LEHD)-N'-methoxycarbonyl-Rhodamine 1 10,
- SEQ ID ⁇ O:3 N-(Ac-WEHD)-N'-methoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O: l N-(Ac-YVAD)-N '-methoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:2 N-(Ac-DEVD)-N '-methoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-methoxycarbonyl-Rhodamine 1 10.
- N-(Ac-DEHD)-N '-methoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-methoxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ O:4 N-(Ac-LEVD)-N '-methoxycarbonyl-Rhodamine 110
- SEQ ID ⁇ O:9 N-(Ac-LEHD)-N'-methoxycarbonyl-Rhodamine 1 10.
- N-(Ac-VEHD)-N'-methoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ N-(Ac-IEPD)-N '-methoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:23 N-(Z-WEHD)-N'-ethoxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O: l N-(Z-YVAD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-ethoxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-ethoxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l N-(Ac-YVAD)
- N-(Ac-LEHD)-N'-octyloxycarbonyl-Rhodamine 1 10 SEQ ID ⁇ O:3 N-(Ac-LETD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 8 N-(Ac-VEHD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:23 N-(Z-WEHD)-V'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:l
- SEQ ID ⁇ O:2 V-(Z-DEVD)-N'-decyloxycarbonyl-Rhodamine 110
- SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-decyloxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-decyloxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ O:l N-(Ac-YVAD)-N'-decyloxycarbonyl-Rhodamine 1 10.
- N-(Ac-DEVD)-N'-decyloxycarbonyl-Rhodamine 1 10 SEQ ID ⁇ O:5 N-(Ac-DEHD)-N ' -decyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-decyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:4 N-(Ac-LEVD)-N'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:9 N-(Ac-LEHD)-N'-decyloxycarbonyl-Rhodamine 1 10. SEQ ID ⁇ O:3
- SEQ ID NO: 8 N-(Ac-VEHD)-N ' -decyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-decyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:23 N-(Z-WEHD)-N'-dodecyloxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:l N-(Z-YVAD)-N'-dodecyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:2
- SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-dodecyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-dodecyloxycarbonyl-Rhodamine 1 10,
- N-(Z-WEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10 SEQ ID ⁇ O: l N-(Z-YVAD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:5 N-(Z-LEVD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ O:9 N-(Ac-WEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID NO: 1
- SEQ ID NO: 8 N-(Ac-VEHD)-N'-(hexylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ N-(Ac-IEPD)-N'-(hexylthio)carbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:2 N-(Z-DEVD)-N'-(octylthio)carbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:6 N-(Ac-DETD)-N'-(octylthio)carbonyl-Rhodamine 110
- SEQ ID ⁇ O:2 N-(Z-LE(OAM)HD(OAM))-N '-acetyl-Rhodamine 1 10
- SEQ ID ⁇ O:3 N-(Z-D(OAM)E(OAM)TD(OAM))-N'-acetyl-Rhodamine 1
- SEQ ID ⁇ O:5 N-(Z-D(OMe)E(OMe)VD(OAM))-N '-acetyl-Rhodamine 1 10.
- SEQ ID NO: 5 N-(Z-D(OMe)E(OMe)VD(OAM))-N '-acetyl-
- N-(Z-D(OMe)E(OMe)VD)-N '-acetyl-Rhodamine 1 10.
- SEQ ID NO: 5 N-(Z-VD(OAM))-N '-acetyl-Rhodamine 1 10, and N-(Z-E(OAM)VD(OAM))-N '-acetyl-Rhodamine 110.
- Another preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula VIII and include, but are not limited to:
- N-(Z-WEHDG)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ OJ1 N-(Z-YVADG)-N '-acetyl-Rhodamine 110, SEQ ID ⁇ OJ2 N-(Z-LEHDG)-N '-acetyl-Rhodamine 1 10.
- SEQ ID ⁇ OJ3 N-(Z-LEVDG)-N '-acetyl-Rhodamine 1 10.
- SEQ ID NO: 74 N-(Z-DETDG)-N '-acetyl-Rhodamine 1 10, SEQ ID ⁇ OJ5
- N-(Z-DEVDG)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-LETDG)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ OJ7 N-(Ac-LEHDG)-N'-acetyl-Rhodamine 110, SEQ ID ⁇ OJ3 N-(Ac-WEHDG)-N'-methoxycarbonyl-Rhodamine 110, SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-methoxycarbonyl-Rhodamine 110, SEQ ID NO: 72
- N-(Ac-DEVDG)-N '-methoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N '-methoxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ OJ8 N-(Z-WEHDGG)-N'-ethoxycarbonyl-Rhodamine 110
- SEQ ID ⁇ OJ9 N-(Z-YVADG)-N'-ethoxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ OJ2 N-(Z-DEVDG)-N'-ethoxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Z-LEVDG)-N'-ethoxycarbonyl-Rhodamine 110
- SEQ ID ⁇ O.J4 N-(Ac-WEHDG)-N'-ethoxycarbonyl-Rhodamine 110
- N-(Ac-WEHDG)-N'-hexyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-hexyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ2 N-(Ac-DEVDG)-N'-hexyloxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N'-hexyloxycarbonyl-Rhodamine 1 10
- SEQ ID ⁇ OJ8 N-(Ac-WEHDG)-N'-octyloxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ OJ 1 SEQ ID ⁇ OJ 1
- N-(Ac-DEVDG)-N'-(decylthio)carbonyl-Rhodamine 110 SEQ ID ⁇ OJ6 N-(Ac-DEHDG)-N'-(decylthio)carbonyl-Rhodamine 110, SEQ ID ⁇ OJ8 N-(Ac-WEHDG)-N'-(dodecylthio)carbonyl-Rhodamine 1 10, SEQ ID ⁇ OJ1 N-(Ac-YVADG)-N'-(dodecylthio)carbonyl-Rhodamine 110, SEQ ID ⁇ OJ2 N-(Ac-DEVDG)-N '-(dodecylthio)carbonyl-Rhodamine 1 10, SEQ ID NO : 76
- Another preferred fluorogenic or fluorescent substrates of the present invention are compounds having Formula IX and include, but are not limited to:
- N-(GPG)-N'-octyloxycarbonyl-Rhodamine 110 N-(GP)-N'-ethoxycarbonyl-Rhodamine 110,
- N-(MGG)-N'-octyloxycarbonyl-Rhodamine 110 N-(MGA)-N ' -octyloxycarbonyl-Rhodamine 1 10
- N-(MA)-N '-ethoxycarbonyl-Rhodamine 110 N-G-N '-ethoxycarbonyl-Rhodamine 110,
- N-(MG)-N'-(ethylthio)carbonyl-Rhodamine 110 N-G-N'-(ethylthio)carbonyl-Rhodamine 110,
- N-(Ac-LM)-N '-ethoxycarbonyl-Rhodamine 110 N-(Boc-LM)-N'-hexyloxycarbonyl-Rhodamine 110, N-(Ac-LM)- ⁇ r -hexyloxycarbonyl-Rhodamine 1 10, N-(Boc-LM)-N'-(ethylthio)carbonyl-Rhodamine 110, N-(Ac-LM)-N'-(ethylthio)carbonyl-Rhodamine 110, N-(Ac-SL ⁇ FPIV)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:80 N-(Ac-SLNFPI)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:81 N-(Ac-SLNFP)-N'-octyloxycarbonyl-Rho
- N-(Ac-IRGGG)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:97 N-(Ac-LVGGG)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:98 N-(Ac-MVGGG)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:99 N-(Ac-IVGGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 100 N-(Ac-LRGGG)-N '-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 101
- N-(Ac-LRGGA)-N '-octyloxycarbonyl-Rhodamine 1 10.
- SEQ ID NO: 102 N-(Ac-LRGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:55 N-(Z-LRGGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:101 N-(Z-LRGGA)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 102 N-(Z-LRGG)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:55
- N-(Ac-LRGGG)-N'-ethoxycarbonyl-Rhodamine 1 10.
- SEQ ID NO: 101 N-(Ac-LRGGA)-N'-ethoxycarbonyl-Rhodam ⁇ ne 1
- SEQ ID NO: 102 N-(Ac-LRGG)-N'-ethoxycarbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:55 N-(Ac-LRGGG)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
- SEQ ID NO: 101 N-(Ac-LRGGA)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
- SEQ ID NO: 102 N-(Ac-LRGGA)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
- N-(Ac-LRGG)-N'-(ethylthio)carbonyl-Rhodamine 1 10.
- SEQ ID ⁇ O:55 N-(Ac-LVLASSS)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 103 N-(Ac-LVLASS)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 104 N-(Ac-LVLAS)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID NO: 105 N-(Ac-LVLA)-N -octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:62
- SEQ ID NO:l 10 N-(Z-Tbg-Tbg-NASS)-A'-octyloxycarbonyl-Rhodamine 110
- SEQ ID NO: 108 N-(Z-Tbg-Tbg-NAS)-A '-octyloxycarbonyl-Rhodamine 1
- SEQ ID ⁇ O:110 N-(Ac-Tbg-Tbg-NASS)-N'-ethoxycarbonyl-Rhodamine 110
- SEQ ID NO: 109 N-(Ac-Tbg-Tbg-NA)-A '-ethoxycarbonyl-Rhodamine 110 SEQ ID NO: 1 10 N-(Ac-Tbg-Tbg-NASS)-N'-(ethylthio)carbonyl-Rhodamine 1 10
- SEQ ID NO: 109 N-(Ac-Tbg-Tbg-NA)-A'-(ethylthio)carbonyl-Rhodamine 110 SEQ ID NO: 110
- SEQ ID ⁇ O:l 1 1 N-(Ac-DIVPCSMST)-V'-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 12 N-(Ac-IVPCSMST)-N ' -octyloxycarbonyl-Rhodamine 110
- SEQ ID ⁇ O:114 N-(Ac-IVPCSM)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID NO: 115
- N-(Ac-IVPCS)-N '-octyloxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 16 N-(Ac-IVPC)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:69 N-(Z-IVPCSMST)-N'-octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l 13 N-(Z-IVPCSMS)-N'-octyloxycarbonyl-Rhodamine 1 10, SEQ ID ⁇ O:l 14 N-(Z-IVPCSM)-N '-octyloxycarbonyl-Rhodamine 1 10, SEQ ID NO: 115 N-(Z-IVPCS)-N -octyloxycarbonyl-Rhodamine 110, SEQ ID ⁇ O:l 16
- N-(Ac-IVPCS)-N'-ethoxycarbonyl-Rhodamine 110 SEQ ID ⁇ O:l 16 N-(Ac-IVPCSMS)-N'-(ethylthio)carbonyl-Rhodamine 110, SEQ ID NO: 114
- Z is benzyloxycarbonyl
- BOC is tert.-butoxycarbonyl
- Ac is acetyl
- Tbg is t-butylglycine
- AM is acetoxymethyl.
- Preferred novel fluorescent dyes of the present invention are compounds having Formula VI and include, but are not limited to:
- N-hexanoyl-Rhodamine 110 N-octanoyl-Rhodamine 110,
- N-ethoxycarbonyl-Rhodamine 110 N-butoxycarbonyl-Rhodamine 110,
- N-dodecyloxycarbonyl-Rhodamine 110 N-benzyloxycarbonyl-Rhodamine 110,
- N-decanesulfonyl-Rhodamine 110 N-dodecanesulfonyl-Rhodamine 110,
- N-trifluoromethanesulfonyl-Rhodamine 116 N-octanesulfonyl-Rhodamine 116,
- Typical aryl groups are C 6 . I0 aryl groups including phenyl, naphthyl, fluorenyl and the like, any of which may be substituted with halo or alkyl groups.
- Typical alkyl groups are C,_ 10 alkyl groups including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and branced chain isomers thereof.
- Typical acyl (alkanoyl) groups are C 2 .. 0 alkanoyl groups such as acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and the like as well as the branched chain isomers thereof.
- Typical biologically acceptable salts of the compounds of the invention include the sodium, potassium, ammonium, TRIS and the like.
- Certain of the compounds of the present invention may be in tautomeric forms, particularly in the y-portion of Formula I.
- the invention includes all such tautomers.
- the invention also includes stereoisomers, the racemic mixtures of such stereoisomers as well as the individual entantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
- the compounds of this invention may be prepared using methods known to those skilled in the art. Specifically, compounds with Formulae I-III can be prepared as illustrated by exemplary reactions in Schemes 1 -5.
- Scheme 1 is the least preferred method since deprotection with HBr/HOAc led to the removal of both the t-butoxy and benzyloxycarbonyl (Z) groups, which makes the next coupling reaction complicated. Thus, where the t-butoxy group is desired, it must be reintroduced.
- N-(9-fluorenylmethoxycarbonyl) (fmoc) group is employed as the N-blocking group (Scheme 2), it can be selectively removed with morpholine, piperidine or other amine base without removing the t-butoxy protecting groups, thus allowing for the ready introduction of additional Z-blocked amino acids or peptides (see Schemes 2-4).
- the final Z- blocked compounds can be selectively deprotected with trifluoroacetic acid (TFA) to remove the t-butoxy group without removing the Z group.
- TFA trifluoroacetic acid
- the invention also relates to a method for the preparation of a compound of Formula III, comprising
- -(AA) ⁇ is WEH, YVA, LEH, DET, DEV, DEH, VEH, LET. SHV, DEL, DGP, DEP, DGT, DL ⁇ , DEE, DSL, DVP, DEA, DSY, ELP, VED, IEP or IET.
- the amino acid is substituted by a carboxy group, it is protected with a OBu-t protecting group which is removed in the final step.
- the condensation reaction may be carried out using any conventional condensing agent that is used for peptide synthesis.
- the condensing agent is l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), or 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ).
- the solvent for the reaction may be pyridine or dimethylformamide (DMF).
- the reaction is generally carried out at room temperature.
- the Fmoc group is generally removed by treatment with morpholine, piperidine or other amine base, in a polar aprotic solvent such as DMF.
- a polar aprotic solvent such as DMF.
- the morpholine is added in excess, and the reaction is carried out at room temperature.
- ⁇ , ⁇ -Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz) is another N- blocking group which can be used in the place of fmoc.
- Ddz-L-aspartic acid ⁇ -t-butyl ester can be used in place of N-fmoc-L-aspartic acid ⁇ -t-butyl ester.
- Ddz can be cleaved selectively in the presence of t-butoxy group by 1 %TFA in methylene chloride.
- the OBu-t group is removed with trifluoroacetic acid in an aprotic solvent such as methylene chloride at room temperature.
- the invention also relates to a method for the preparation of a compound of Formula VII, comprising
- -(AA) repeat is WEH.
- Compounds of Formula VII also can be prepared using an acyl (alkanoyl) chloride in place of acetic anhydride, such as acetyl chloride, hexanoyl chloride, octanoyl chloride and decanoyl chloride.
- acyl alkanoyl
- reagents can be used in place of acetic anhydride include, but are not limited to carbamyl chloride such as dimethylcarbamyl chloride, diethylcarbamyl chloride and N-methyl-N- hexylcarbamyl chloride; chloroformate such as methyl chloroformate, ethyl chloroformate, octyl chloroformate, 2-butoxyethyl chloroformate and 2,5,8- trioxadecyl chloroformate; chlorothiolformate such as methyl chlorothiolformate, ethyl chlorothiolformate, octyl chlorothiolformate; alkyl, haloalkyl and aralkyl sulfonyl halides such as methanesulfonyl chloride, octanesulfonyl chloride, trifluoromethanesulfonyl chloride and tosylchloride.
- the reaction is carried out in the presence of a base, such as (Et) 3 ⁇ , (i-Pr) 2 -NEt or pyridine.
- a base such as (Et) 3 ⁇ , (i-Pr) 2 -NEt or pyridine.
- the preferred solvent is DMF.
- the reaction is generally carried out at room temperature.
- the ratio of anhydride or acyl chloride to Rhodamine is about 1 :1.
- the condensation reaction may be carried out using any conventional condensing agent that is used for peptide synthesis.
- the condensing agent is EDC or EEDQ
- the solvent for the reaction is pyridine or dimethylformamide (DMF).
- the reaction is generally carried out at room temperature.
- the ratio of condensing agent to N-acetyl-Rhodamine is about 3:1 and the ratio of protected amino acid or peptide to N-acetyl-Rhodamine or N- (Asp(OBu-t))-N'-acetyl-Rhod.amine is about 3:1.
- compounds of Formula VI provide fluorescent dyes which can be condensed with any peptide or other structure for the preparation of fluorogenic or fluorescent compounds which are substrates for proteases or peptidases.
- compounds of Formula VII also can be prepared by first condensing a peptide with a Rhodamine to give N-peptide-Rhodamine, then reacting the N-peptide-Rhodamine with acetyl anhydride or other acylating reagent to give for example, N-acetyl-N'-peptide-Rhodamine.
- a) peptides in general are much more expensive than acyl chlorides or anhydrides
- the condensation reaction between peptide and Rhodamine is not an efficient reaction. For these reasons it is preferred to attach the peptide to N-acetyl- Rhodamine rather than attach the acyl group to N-peptide-Rhodamine.
- the invention relates to a method for determining whether a test substance has an effect on an enzyme involved in the apoptosis cascade in a test cell, comprising
- test substance either interacts with an external membrane receptor of the cell or is taken into the cell and the reporter compound is taken into the cell
- Activators either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade include but are not limited to known chemotherapeutic agents, such as etoposide (Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK (1998), Biochim Biophys Ada 1395:110-120) and doxorubicin (Gamen S, Anel A, Lasierra P, Alava MA, Martinez-Lorenzo MJ, Pineiro A, Naval J (1997), FEBS
- chemotherapeutic agents such as etoposide (Yoon HJ, Choi IY, Kang MR, Kim SS, Muller MT, Spitzner JR, Chung IK (1998), Biochim Biophys Ada 1395:110-120) and doxorubicin (Gamen S, Anel A, Lasierra P, Alava MA, Martinez-Lorenzo MJ, Pineiro A, Naval J
- Inactivators either by direct or indirect mechanisms, of enzymes involved in the apoptosis cascade include but are not limited to endogenous proteins including Bcl-2 (Joensuu H, Pylkkanen L, Toikkanen S (1994;, Am. J. Pathol 5: 1 191- 1198), the viral produced agent p35 (Miller LK (1997;, J. Cell Physiol 173: 178-
- the invention relates to the use of the reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays, using whole cells or tissue samples which have been induced to undergo apoptosis, to screen for compounds that inhibit either directly or indirectly an enzyme or enzymes involved in apoptosis (programmed cell death).
- These screening assays using compounds having Formulae I-III, V, VII and VIII are expected to lead to discovery of new drugs or new uses for known drugs that slow or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.
- the reporter compounds having Formulae I-III, V, VII and VIII and the screening assays of the present invention can be used to identify drugs that reduce or prevent cell death in the nervous system (brain, spinal cord, and peripheral nervous system) under various conditions of ischemia and excitotoxicity, including, but not limited to, focal ischemia due to stroke and global ischemia due to cardiac arrest.
- the screening assays can also be used to identify compounds that reduce or prevent cell death in the nervous system due to traumatic injury (such as head trauma or spinal cord injury), viral infection or radiation-induced nerve cell death (for example, as a side-effect of cancer radiotherapy) or environmental toxicity (e.g. by certain halogenated hydrocarbon).
- the screening assays can also be used to identify cell death inhibitors which are useful to reduce or prevent cell death in a range of neurodegenerative disorders, including but not limited to Alzheimer's disease, Huntington's Disease, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, and spinobulbar atrophy.
- the screening assays of this invention can be used to identify compounds that prevent cell death in any condition which potentially results in the death of cardiac muscle. This includes myocardial infarction, congestive heart failure and cardiomyopathy.
- One particular application of the screening assay is to identify compounds which reduce or prevent myocardial cell death that occurs in certain viral infections of the heart.
- the screening assays of the invention can be used to identify compounds which prevent cell death of retinal neurons that occurs in disorders associated with increased intraocular pressure (such as glaucoma) or retinal disorders associated with the aging process (such as age-related macular degeneration).
- the assays can also be used to identify compounds which treat hereditary degenerative disorders of the retina, such as retinitis pigmentosa.
- the screening assays of the invention can also be used to identify cell death inhibitors that can be used to reduce or prevent premature death of cells in the immune system, and are particularly useful in identifying inhibitors which are useful in treating immune deficiency disorders, such as acquired immune deficiency syndrome (AIDS), severe combined immune deficiency syndrome (SCIDS) and related diseases.
- the screening assays can also be used to identify cell death inhibitors that can be used to treat radiation-induced immune suppression.
- the screening assays of the invention can also be used to identify drugs useful in organ transplantation procedures. Transplantation of human organs and tissues is a common treatment for organ failure. However, during the transplantation process, the donor organ or tissue is at risk for cell death since it is deprived of its normal blood supply prior to being implanted in the host. This ischemic state can be treated with cell death inhibitors by infusion into the donor organ or tissue, or by direct addition of the cell death inhibitors to the organ/tissue storage medium. Such cell death inhibitors can be identified using the screening assays described in this invention. Cell death inhibitors may also be used to reduce or prevent cell death in the donor organ/tissue after it has been transplanted to protect it from the effects of host immune cells which kill their targets by triggering apoptosis.
- the screening assays described in this invention can be used to identify cell death inhibitors useful in protecting transplanted organs from rejection.
- the cytoprotective effects of cell death inhibitors can also be used to prevent the death of human or animal sperm and eggs used in in vitro fertilization procedures. These inhibitors can be used during the harvesting process and can also be included in the storage medium.
- Cell death inhibitors useful for application in fertilization procedures can be identified using the screening assay methods described in this invention.
- Mammalian cell lines and yeast cells are commonly used to produce large amounts of recombinant proteins (such as antibodies, enzymes or hormones) for industrial or medicinal use.
- the lifespan of some of these cell lines is limited due to growth conditions, the nature of the recombinant molecule being expressed (some are toxic) and other unknown factors.
- the lifespans of industrial cell lines can be extended by including cell death inhibitors in the growth medium.
- Cell death inhibitors useful in extending the life span of cell lines can be identified using the screening assay procedures described in this invention.
- hair follicle regression (referred to as catagen) may be due at least partially to apoptosis. Therefore, it is possible that cell death inhibitors can be used to treat hair loss that occurs due to various conditions, including but not limited to male-pattern baldness, radiation-induced or chemotherapy-induced hair loss, and hair loss due to emotional stress. There is also evidence that apoptosis may play a role in the loss of hair color. Therefore, it is possible that cell death inhibitors can also be used in treating cases of premature graying of the hair. Cell death inhibitors useful in treating or preventing hair loss or graying of the hair can be identified using the screening assay procedures described in this invention.
- the death of skin epithelial cells can occur after exposure to high levels of radiation, heat or chemicals. It is possible that cell death inhibitors can be used to reduce or prevent this type of skin damage. In one particular application, cell death inhibitors can be applied in an ointment to treat acute over-exposure to the sun and to prevent blistering and peeling of the skin. Cell death inhibitors useful in treating or preventing death of skin cells can be identified using the screening assay procedures described in this invention.
- reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays using live or dead whole cells or tissue samples to screen for compounds that stimulate, either directly or indirectly, an enzyme or enzymes involved in apoptosis.
- reporter compounds having Formulae I-III, V, VII and VIII in whole-cell assays using yeast and other fungi, and bacteria to screen compound libraries for anti-fungal or anti-bacterial drugs that act by inducing, either directly or indirectly, the caspase cascade or other enzymes involved in apoptosis in those cells.
- Another important aspect of the invention is to use the reporter compounds having Formulae I-III, V, VII and VIII to monitor the therapeutic effects of therapeutic agents or treatments given to patients with the aim of reducing, preventing or treating maladies in which apoptotic cell death is either a cause or a result.
- Another important aspect of the present invention is to use the reporter compounds having Formulae IX to screen for HIV protease inhibitors in HIV infected cells, comprising
- test substance either interacts with an external membrane receptor or is taken into said cell and the reporter compound is taken into the cell
- Yet another important aspect of the present invention is to use the reporter compounds having Formulae IX to diagnose HIV infection, comprising (a) contacting a test cell from an individual suspected of having HIV infection with the reporter compound according to the invention under conditions whereby the reporter compound is taken into the cell, and
- the reporter compounds having Formula IX of the present invention can be used to screen for adenovirus protease inhibitors in adenovirus infected cells.
- the reporter compounds having Formula IX of the present invention also can be used to screen for herpes simplex virus type-1 (HSV-1) protease inhibitors in HSV-1 infected cells.
- HSV-1 herpes simplex virus type-1
- the reporter compounds also can be used to screen for human cytomegalovirus (HCMV) protease inhibitors in HCMV infected cells; to screen for hepatitis C virus (HCV) protease inhibitors in HCV infected cells; to screen for DPP-IV inhibitors in T-cells; as well as to screen for type-2 methionine aminopeptidase (MetAP-2) inhibitors in endothelial cells.
- HCMV human cytomegalovirus
- HCV hepatitis C virus
- MethodAP-2 type-2 methionine aminopeptidase
- the reporter compounds having Formula IX of the present invention also can be used to diagnose adenovirus, herpes simplex virus type-1, human cytomegalovirus and hepatitis C virus.
- compositions within the scope of this invention include all compositions wherein the fluorogenic or fluorescent compounds of the present invention are contained in an amount which are effective to achieve its intended purpose.
- the fluorogenic or fluorescent substrate compounds may be applied to cells or cell lines from mammals, e.g. humans, or other animals by incubating the cells or tissues containing the cells with the fluorogenic or fluorescent substrate at a concentration of about 0.01 nanomolar to about 1 molar, or an equivalent amount of a salt or proreporter molecule thereof in a physiologically compatible buffer.
- buffers include cellular growth medias, an example for leukemia derived cancer cells being RPMI-1640 with or without 10% fetal bovine serum.
- Other known cellular incubation buffers could involve isotonic solutions buffered with either phosphate or HEPES.
- the cells can be derived from any organ or organ system for which it is desirable to find— by- means of the screening assays— drugs that could be useful in treating apoptosis- mediated disorders, e.g., neuronal cell death, heart disease, liver disease, retinal disorders, kidney, joint and bone diseases, immune system disorders, cancers, tumors and tissue hyperplasias etc.
- apoptosis- mediated disorders e.g., neuronal cell death, heart disease, liver disease, retinal disorders, kidney, joint and bone diseases, immune system disorders, cancers, tumors and tissue hyperplasias etc.
- Suitable solubilizers may be used for presenting the fluorogenic or fluorescent compounds of the present invention to tissues, cells or cell lines.
- Suitable solubilizers include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts and alkaline solutions.
- suspensions of the compounds as appropriate oily suspensions may be presented to the cells or tissues.
- Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400) or dimethylsulfoxide (DMSO) or another suitable solubilizer.
- the invention also relates to the pro-reporter derivatives of the compounds of the invention.
- pro-reporter derivatives include compounds which are cleaved in situ by endogenous enzymes to give the compounds of Formulae I-III, V, and VII-IX.
- Such pro-reporter derivatives include lower alkyl esters of carboxyl-containing amino acid residues such as Asp and Glu.
- Especially preferred pro-reporter derivatives include the methyl esters and acetoxymethyl (AM) esters of Asp- and Glu-containing compounds.
- the following examples demonstrate usefulness of the invention in measuring the activity of caspases and other enzymes involved in apoptosis in cells and tissues.
- Rhodamine 110 500 mg, 1.36 mmol
- DMF dimethyl methoxysulfoxide
- acetic anhydride 167 mg. 1.64 mmol was added dropwise to the above solution.
- the reaction solution was stirred at room temperature for 24 h. and it was then diluted with 100 mL of water and extracted with ethyl acetate (3 x 50 mL).
- Rhodamme 1 10 (3.00 g, 8.18 mmol) dissolved in dimethylformamide (140 mL) at -50 °C was added N,N-diisopropylethylamine (1.27 g, 1.2 mmol), then ethyl chloroformate (1.07 g, 9.81 mmol) was added dropwise to the above solution.
- the reaction solution was then slowly warmed to room temperature and kept stirring for 5 h. It was then diluted with 200 mL of ice water and extracted with ethyl acetate (3 x 50 mL).
- Rhodamine 110 SEQ ID ⁇ O:5
- Rhodamine 110 SEQ ID ⁇ O:5
- Rhodamine 1 16 (458.8 mg, 1.0 mmol), NN-diisopropylethylamine (129.3 mg, 1.0 mmol) and acetic anhydride (122 mg, 1.2 mmol) (as described in
- Example 25 was obtained 141 mg (9.4%>) of the title compound as colorless solid.
- ⁇ ⁇ MR (CDC1 3 ): ⁇ 8.01 (d, J 7.5 Hz, IH), 7.69-7.62 (m, 3H), 7.24-6.36 (m. 7H), 3.95 (bs, 2H), 3.28 (s, 3H), 2.87 (s, 3H), 1.95 (bs, 3H).
- Rhodamine 110 500 mg, 1.36 mmol
- dimethylformamide (12 mL) at -61 °C NN-diisopropylethylamine (264 mg, 2.04 mmol)
- ethyl chlorothiolformate 204 mg, 1.64 mmol
- the reaction solution was then slowly warmed to room temperature and kept stirring for 1 h. It was then diluted with 100 mL of ice water and extracted with ethyl acetate (3 x 30 mL).
- N-ethoxycarbonyl Rhodamine 110 (58 mg, 0.135 mmol) (according to Example 1) was obtained the title compound (70 mg, 83%) as a solid.
- Rhodamine 1 10 0.5 g, 1.36 mmol
- DMF 15 ml
- NN-diisopropylethylamine 0.25 ml
- N-hexyl-N- methylcarbamyl chloride in DMF (2.05 mmol.
- the reaction mixture was stirred at -61°C for 1 h, then was allowed to warm up to rt.
- the reaction mixture was further stirred at rt for 14 h, and then was partitioned between aqueous saturated ⁇ H 4 C1 solution and ethyl acetate (2 x 50 ml).
- Rhodamine 110 and N-acetyl-Rhodamine 110 were measured in a fluorometric assay.
- the fluorescent signal is read in a spectrofluorometer or in a fluorometric microtiter plate reader at excitation wavelength of 485 and emission 530. Using this assay, the relative fluorescent values were determined for the two fluorescent moieties.
- Rhodamine 1 10
- N-acetyl-Rhodamine 110 is a stable and efficient fluorescent indicator.
- Modified Rhodamine dyes were evaluated using both conventional spectrometry and spectrofluorometry. For both types of analysis the dyes were dissolved in either methanol or 50 mM Tris at final dye concentrations ranging from 10 nM to 100 ⁇ M. An absorbance spectrum from wavelengths of 200 ran to 700 nm was determined for each dye using a Beckman DU-7000 spectrophotometer. The dyes all had absorbance peaks at around 470 to 480 nm. This wavelength was chosen as the fluorescence excitation wavelength and a full fluorescence emission spectrum was determined using a Hitachi F-2000 spectrofluorometer. For each dye, the emission peak was around 520 nm and the fluorescent output was measured under the conditions tested (see Table 1).
- HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 ⁇ M or 50 ⁇ M N-octyloxycarbonyl-Rhodamine 110, N- decyloxycarbonyl-Rhodamine-110, N-dodecyloxycarbonyl-Rhodamine-110, N- hexyloxycarbonyl-Rhodamine-110, N-(ethylthio)carbonyl-Rhodamine 110 or Rhodamine 110.
- the cells were incubated for varying times at 37°C in a C0 2 incubator, recovered by centrifugation, and washed in 50 mL of ice-cold medium.
- N-octyloxycarbonyl-Rhodamine 1 10 (Fig. 1A), N- decyloxycarbonyl-Rhodamine 1 10 (Fig. IB), and N-dodecyloxycarbonyl- Rhodamine 110 (Fig. 1C) stained HL-60 cells intensely and there was almost no leakage of the dye into the medium.
- N-Hexyloxycarbonyl-Rhodamine 1 10 (Fig.
- Rhodamine 1 10 (Fig. IF) stained cells rapidly, but the dye quickly leaked out of the cells, resulting in a low intensity of cellular staining and a high degree of fluorescence in the medium containing the cells. Therefore, the modified Rhodamine dyes are superior to Rhodamine 110 since they are readily taken up by HL-60 cells and are retained within the cells for at least 30 minutes.
- N-(Z-VD)-N '-acetyl-Rhodamine 110 The activities of N-(Z-VD)-N '-acetyl-Rhodamine 110, N-(Z-VAD)-N'- acetyl-Rhodamine 110, N-(Z-DEVD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:5,
- N-(Z-YVAD)-N-acetyl-Rhodamine 110 SEQ ID ⁇ O:2, (Z-VAD) 2 -Rhodamine 110 and (Z-YVAD) 2 -Rhodamine 110 SEQ ID NO:2 as synthetic substrates for recombinant CPP32 and ICE were measured in a fluorometric enzyme assay.
- Recombinant CPP32 protein and ICE protein were prepared by expressing DNA clones encoding these enzymes in an insect host cell (sf9 cells) using baculovirus as the vector. See. Webb, N.R. et al. "Expression of proteins using recombinant Baculovirus," Techniques 2:173-188 (1990).
- CPP32 and ICE dependent substrate cleavage was measured using the following buffer conditions: 100 mM HEPES pH 7.5, with 10% sucrose, 1% CHAPS, 5 mM glutathione, and 1-100 ⁇ M test substrate.
- Nonspecific enzyme cleavage was determined with the use of the specific CPP32 and ICE inhibitors consisted of an oligomer with the sequence Asp-Glu- Val-Asp or Tyr-Val-Ala-
- the assay for enzyme activity was typically carried out at 37°C for 60 minutes.
- Table 4 lists the K_- and V max values for N-(Z-VD)-N '-acetyl-Rhodamine 1 10, N-(Z-VAD)-N '-acetyl-Rhodamine 110, N-(Z-DEVD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:5, N-(Z-YVAD)-N '-acetyl-Rhodamine 110 SEQ ID ⁇ O:2, (Z-
- N-(Z-VD)-N'-Ac-Rhodamine 110 60 1 1 NA N-(Z-VAD)-N'-Ac-Rhodamine 1 10 NA 70 4 N-(Z-DEVD)-N'-Ac-Rhodamine 1 10 154 160 12 9 SEQ ID ⁇ O:5
- SEQ ID NO:2 NA no activity observed at 1- 100 ⁇ M substrate, 37°C, 3 h incubation
- N-(Z-DEVD)-N '-acetyl- Rhodamine 110 SEQ ID ⁇ O:5 is an efficient substrate for both ICE and CPP32. Also shown is that N-(Z-VD)-N '-acetyl-Rhodamine 1 10 is an efficient substrate for CPP32 and not for ICE and that N-(Z-VAD)-N '-acetyl-Rhodamine 1 10, N-(Z- YVAD)-N '-acetyl-Rhodamine 1 10 SEQ ID ⁇ O:2, (Z-VAD) 2 -Rhodamine 1 10 and (Z-YVAD) 2 -Rhodamine 110 SEQ ID NO:2 are efficient substrates for ICE and not for CPP32.
- the caspase substrates were assayed by recombinant caspase-3 and by lysates prepared from apoptotic HL-60 cells.
- the assays were carried out at 37°C in 96-well plates in a 100 ⁇ L incubation containing 30 ⁇ L of caspase-3 preparation or cell lysate, 10 ⁇ M or 50 ⁇ M of the substrate, and caspase assay buffer (40 mM 1 ,4-piperazinebis(ethansulfonic acid) (PIPES, Aldrich Chemical Company) pH 7.2; 100 mM NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT).
- caspase assay buffer 40 mM 1 ,4-piperazinebis(ethansulfonic acid)
- N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 was tested using apoptotic HL-60 and Jurkat cells. These whole-cell assays were carried out in two stages: 1) induction of apoptosis; 2) incubation with the substrate. For HL-60 cells, apoptosis was induced by treatment with 10 ⁇ g/ml vinblastine for 4 hours. Control samples were treated with DMSO. For Jurkat cells, apoptosis was induced by treatment with 500 ng/ml agonistic antiFas antibody for 2 hours. Control samples were treated with PBS.
- the cells were incubated with 50 ⁇ M N-Ac-DEVD-N-octyloxycarbonyl-Rl lO SEQ ID ⁇ O:5 in caspase assay buffer (40 mM PIPES, pH 7.4; 100 mM NaCl; 10% sucrose; 1 mM EDTA; 10 mM DTT).
- caspase assay buffer 40 mM PIPES, pH 7.4; 100 mM NaCl; 10% sucrose; 1 mM EDTA; 10 mM DTT.
- the cells were than transferred to a glass microslide and viewed by epifluorescent illumination on a Nikon inverted microscope.
- vinblastine-treated HL-60 cells were intensely stained by N-Ac-DEVD-N'- octyloxycarbonyl R110 SEQ ID ⁇ O:5.
- DMSO-treated cells also showed some staining (Fig. 3B). Although the intensity of the signal was significantly less than that of vinblastine-treated cells.
- HL-60 cells treated with 50 ⁇ M Ac-DEVD-CHO SEQ ID NO: 5 during the assay stage (Fig. 3C) showed almost no fluorescent signal, indicating that the staining observed in vinblastine-treated cells is almost entirely due to caspase-mediated cleavage.
- Jurkat cells induced to undergo apoptosis by antiFas (Fig. 3D) also showed intense staining by N-Ac-DEVD-N'- octyloxycarbonyl R1 10 SEQ ID ⁇ O:5, while control cells showed only light staining (Fig. 3F).
- N-Ac-DEVD-N'- octyloxycarbonyl Rl 10 SEQ ID ⁇ O:5 can be used to measure apoptosis in intact cells and that the signal obtained from N-Ac-DEVD-N'-octyloxycarbonyl-Rl lO SEQ ID NO: 5 is caspase-dependent.
- N-Ac-LEVD-N-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 was assayed by recombinant human caspase-3, 6, 7, and 8.
- the assays were carried out at 37°C in 96-well plates in a 100 ⁇ L incubation containing recombinant human caspase, 10 ⁇ M of N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9, and caspase assay buffer (40 mM PIPES, pH 7.2; 100 mM NaCl; 10% sucrose; 0.1% CHAPS; 1 mM EDTA; 10 mM DTT).
- caspase-6 and caspase-8 cleave N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 to give an easily measured fluorescent signal (signal to background ratios of about 13 for caspase-6 to about 26 for caspase-8).
- Caspase-3 cleaved N-Ac-LEVD-N'-ethoxycarbonyl Rl lO SEQ ID ⁇ O:9 less efficiently, yielding a signal that was about 5-fold above the enzyme blank value.
- Caspase-7 gave virtually no signal.
- Aminopeptidases are present in many cells and sequentially remove unblocked amino acid residues from peptides, starting from the N-terminus. Peptides with blocked amino termini are not cleaved.
- HL-60 lysates were prepared by homogenizing HL-60 cells in caspase buffer, and the ability of these lysates to cleave N-Z-G-N'-octyloxycarbonyl Rl 10 and N-G-N'-octyloxycarbonyl- Rl lO was tested in a microtiter plate assay.
- Figure 6 shows that HL-60 cell lysates readily cleaved N-G-N'-octyloxycarbonyl Rl l O, and the size of the signal was dependent on the concentration of substrate. By contrast, no signal was generated by HL-60 cell lysates from N-Z-G-N'-octyloxycarbonyl-Rl 10.
- Drugs that stimulate the caspase cascade in the absence of Fas ligand may be useful, for example, as anti-cancer chemotherapeutic agents.
- the assay described in Example 78 may be used to screen for drugs that stimulate the caspase cascade by carrying out the assay under similar conditions as in Example 78, except that a known or unknown compound with known or unknown anti- cancer or anti-tumor activity replaces the Fas ligand reagent.
- fluorescence assay in screening for drugs that inhibit or potentiate the caspase cascade stimulated with Fas ligand or another apoptosis inducer.
- the assays and reagents described in this invention may be used to screen for drugs that either inhibit or potentiate the caspase cascade in cells by performing the assay as described in Example 78 using Fas ligand or any other agent that stimulates the caspase cascade or other apoptosis pathway in the presence of a test substance that inhibits or potentiates or acts synergistically with the action of the first apoptosis or caspase cascade inducer.
- the fluorescence assays described in this invention permit chemosensitivity or drug resistance testing of cancer or tumor cells or tissue samples taken from individual cancer or tumor patients.
- a fluorescence assay using a cancer cell or tissue sample taken from a patient may be conducted as described Example 78.
- different drugs with known or unknown chemotherapeutic activity can be tested for their capacity to stimulate the caspase cascade.
- the results from this assay provide information that can be used to design an optimal chemotherapeutic drug treatment regimen for the patient.
- HL-60 cells were placed in 5 ml of Iscove's medium (without serum or phenol-red) containing 10 ⁇ M N-G-N -octyloxycarbonyl-Rl 10 or 10 ⁇ M N-Z-G— N-octyloxycarbonyl-Rl 10.
- Iscove's medium without serum or phenol-red
- Three million HL-60 cells were incubated for 3 hours at 37 °C in a C0 2 incubator, recovered by centrifugation, and washed in 50 ⁇ L of ice-cold medium. The cells were re-centrifuged and the final pellet was resuspended in 50 ⁇ L of fresh medium.
Abstract
Description
Claims
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US33661 | 1979-04-26 | ||
US6158297P | 1997-10-10 | 1997-10-10 | |
US61582P | 1997-10-10 | ||
US3366198A | 1998-03-03 | 1998-03-03 | |
PCT/US1998/021231 WO1999018856A1 (en) | 1997-10-10 | 1998-10-09 | Novel fluorescent reporter molecules and their applications including assays for caspases |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1026988A1 true EP1026988A1 (en) | 2000-08-16 |
EP1026988A4 EP1026988A4 (en) | 2005-03-30 |
Family
ID=26709974
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98953317A Withdrawn EP1026988A4 (en) | 1997-10-10 | 1998-10-09 | Novel fluorescent reporter molecules and their applications including assays for caspases |
Country Status (15)
Country | Link |
---|---|
EP (1) | EP1026988A4 (en) |
JP (1) | JP2001519368A (en) |
KR (1) | KR20010031056A (en) |
CN (1) | CN1281346A (en) |
AU (1) | AU754634B2 (en) |
BR (1) | BR9814816A (en) |
CA (1) | CA2308125A1 (en) |
EA (1) | EA200000408A1 (en) |
HU (1) | HUP0100079A2 (en) |
IL (1) | IL135365A0 (en) |
IS (1) | IS5414A (en) |
NO (1) | NO20001322L (en) |
NZ (1) | NZ503619A (en) |
PL (1) | PL341661A1 (en) |
WO (1) | WO1999018856A1 (en) |
Families Citing this family (49)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6342611B1 (en) | 1997-10-10 | 2002-01-29 | Cytovia, Inc. | Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for capsases and other enzymes and the use thereof |
AU5116099A (en) | 1998-07-21 | 2000-02-14 | Sui Xiong Cai | Novel fluorescence dyes and their applications for whole cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof |
WO2000054049A2 (en) * | 1999-03-12 | 2000-09-14 | Evotec Analytical Systems Gmbh | Determination of the chemosensitivity via the caspase activity |
EP1055929A1 (en) * | 1999-05-26 | 2000-11-29 | Tibotec N.V. | Means and methods for drug discovery and the phenotypic characterisation of cells |
EP1185644A1 (en) * | 1999-05-27 | 2002-03-13 | Merck Frosst Canada & Co. | Assays for caspase activity using green fluorescent proteins |
US6261794B1 (en) | 1999-10-14 | 2001-07-17 | Saint Louis University | Methods for identifying inhibitors of methionine aminopeptidases |
DE60008574T2 (en) | 1999-11-05 | 2009-10-01 | Cytovia, Inc., San Diego | Substituted 4H-chromenes and analogues as caspase activators and apoptosis inducers and their use |
GB9927331D0 (en) | 1999-11-18 | 2000-01-12 | Fluorescience Ltd | Assay for measuring enzyme activity in vivo |
AU2001293372A1 (en) | 2000-04-18 | 2001-10-30 | Cytovia, Inc. | Substituted 1,4-thiazepine and analogs and their use as activators of caspases |
WO2002012545A2 (en) * | 2000-08-03 | 2002-02-14 | Cytovia, Inc. | Method of identifying immunosuppressive agents |
DE10053747C2 (en) * | 2000-10-30 | 2002-10-24 | Deutsches Krebsforsch | Method for the simultaneous determination of two fluorescence emissions with a single laser flow cytometer |
EP1205540A1 (en) | 2000-11-10 | 2002-05-15 | Evotec OAI AG | Method of measuring cell vitality |
WO2002102301A2 (en) | 2000-12-07 | 2002-12-27 | Cytovia, Inc. | Substituted indole-2-carboxylic acid benzylidene-hydrazides and analogs as activators of caspases and inducers of apoptosis and the use thereof |
US6716851B2 (en) | 2000-12-12 | 2004-04-06 | Cytovia, Inc. | Substituted 2-aryl-4-arylaminopyrimidines and analogs as activators or caspases and inducers of apoptosis and the use thereof |
AU2002303123A1 (en) | 2001-03-14 | 2002-09-24 | Cytovia, Inc. | Multifluoro-substituted chalcones and analogs as activators of caspases and inducers of apoptosis and the use thereof |
US6979530B2 (en) | 2001-05-21 | 2005-12-27 | Applera Corporation | Peptide conjugates and fluorescence detection methods for intracellular caspase assay |
US7148030B2 (en) | 2002-02-01 | 2006-12-12 | Promega Corporation | Bioluminescent protease assay |
US7528164B2 (en) | 2002-05-16 | 2009-05-05 | Cytovia, Inc. | Substituted 4-aryl-4h-pyrrolo[2,3-h]chromenes and analogs as activators of caspases and inducers of apoptosis and the use thereof |
WO2003096982A2 (en) | 2002-05-16 | 2003-11-27 | Cytovia, Inc. | Substituted 4h-chromenes, 2h-chromenes, chromans and analogs as activators of caspases and inducers of apoptosis and the use thereof |
CA2491692C (en) * | 2002-07-08 | 2010-12-07 | Amersham Biosciences Uk Limited | Reagents and a method for saturation labelling of proteins |
AU2003299378A1 (en) * | 2002-10-11 | 2004-05-04 | Board Of Regents, The University Of Texas System | Method and compounds for inhibiting hec1 activity for the treatment of proliferative diseases |
US6821744B2 (en) | 2002-10-29 | 2004-11-23 | Roche Diagnostics Operations, Inc. | Method, assay, and kit for quantifying HIV protease inhibitors |
WO2005003100A2 (en) | 2003-07-03 | 2005-01-13 | Myriad Genetics, Inc. | 4-arylamino-quinazolines as activators of caspases and inducers of apoptosis |
WO2005047245A2 (en) | 2003-11-14 | 2005-05-26 | Wisconsin Alumni Research Foundation | Fluorescence assays with improved blocking groups |
EP1704257A2 (en) | 2004-01-16 | 2006-09-27 | Applera Corporation | Fluorogenic kinase assays and substrates for kinases and phosphatases |
US7553632B2 (en) | 2004-01-22 | 2009-06-30 | Promega Corporation | Luminogenic and nonluminogenic multiplex assay |
US7416854B2 (en) | 2004-01-22 | 2008-08-26 | Promega Corporation | Luminogenic and nonluminogenic multiplex assay |
NZ566799A (en) | 2005-09-14 | 2011-04-29 | Takeda Pharmaceutical | Dipeptidyl peptidase inhibitors for treating diabetes |
CN101360723A (en) | 2005-09-16 | 2009-02-04 | 武田药品工业株式会社 | Process for the preparation of pyrimidinedione derivatives |
JP5380074B2 (en) * | 2005-12-12 | 2014-01-08 | イノーバ・バイオサイエンシズ・リミテッド | Complex generation |
WO2007112347A1 (en) | 2006-03-28 | 2007-10-04 | Takeda Pharmaceutical Company Limited | Dipeptidyl peptidase inhibitors |
TW200838536A (en) | 2006-11-29 | 2008-10-01 | Takeda Pharmaceutical | Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor |
US8227205B2 (en) | 2007-04-13 | 2012-07-24 | Promega Corporation | Luminescent live and dead cell assay |
KR100909996B1 (en) * | 2007-09-19 | 2009-07-29 | 한국생명공학연구원 | Fusion protein for detecting the activity of caspase-3 and a method for detecting the activity of caspase-3 using the same |
CN102603695B (en) * | 2012-02-10 | 2014-06-18 | 山东大学 | Amino acid-fluorophore compound and application thereof |
WO2014013272A1 (en) | 2012-07-19 | 2014-01-23 | Kingston University Higher Education Corporation | Rhodamine based fluorescent probe for coagulase activity and detection of coagulase producing bacterial strains |
CN104458709A (en) * | 2013-09-12 | 2015-03-25 | 中国药科大学 | High flux screening method for screening calcium activated neutral protease-1 inhibitor |
ES2806134T3 (en) | 2014-07-11 | 2021-02-16 | Univ Tokyo | Fluorescent probe to detect dipeptidyl peptidase IV |
CN104263356B (en) * | 2014-09-29 | 2016-02-24 | 辽宁大学 | A kind of rhodamine pH fluorescent probe and application thereof containing methionine(Met) structure |
CN106146611B (en) * | 2015-05-14 | 2019-09-17 | 中国科学院大连化学物理研究所 | It is a kind of measure dipeptidyl peptidase IV activity fluorescence probe substrate and its application |
EP3475443A1 (en) | 2016-06-23 | 2019-05-01 | Life Technologies Corporation | Methods and compositions for detecting or measuring caspases or apoptosis |
WO2018071556A1 (en) * | 2016-10-11 | 2018-04-19 | The Regents Of The University Of California | Detection, identification, and purification of degradative and non-degradative enzymes in biological samples |
CN108129388A (en) * | 2018-01-08 | 2018-06-08 | 深圳市佶达德科技有限公司 | A kind of rhodamine double pyrazole acetic acid esters organic laser material and its application |
CN107987019A (en) * | 2018-01-08 | 2018-05-04 | 深圳市佶达德科技有限公司 | A kind of preparation method of rhodamine double pyrazole acetic acid esters organic laser material |
CN108760234B (en) * | 2018-06-05 | 2020-05-15 | 哈尔滨工程大学 | Method and device for synchronously testing fluid flow and solid motion information based on PIV (particle image velocimetry) and PTV (particle beam velocimetry) technologies |
KR102138256B1 (en) * | 2018-09-13 | 2020-08-13 | 한국과학기술연구원 | probe for measuring the activity of caspase-1 and composition for diagnosis of inflammatory diseases comprising the same |
CN112961209B (en) * | 2021-04-07 | 2022-06-24 | 中国科学院武汉病毒研究所 | caspase inhibitors and uses thereof |
CN114107435A (en) * | 2021-11-30 | 2022-03-01 | 广东省人民医院 | Activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy and application |
CN117143949B (en) * | 2023-08-31 | 2024-04-05 | 青岛海洋食品营养与健康创新研究院 | Euphausia superba source high F value oligopeptide and application thereof in liver protection |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996033209A1 (en) * | 1995-04-21 | 1996-10-24 | Merck Frosst Canada Inc. | Peptidyl derivatives as inhibitors of pro-apoptotic cysteine proteinases |
US5576424A (en) * | 1991-08-23 | 1996-11-19 | Molecular Probes, Inc. | Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells |
WO2000004914A1 (en) * | 1998-07-21 | 2000-02-03 | Cytovia, Inc. | Novel fluorescence dyes and their applications for whole cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4557862A (en) * | 1983-10-28 | 1985-12-10 | University Patents, Inc. | Rhodamine derivatives as fluorogenic substrates for proteinases |
US5587490A (en) * | 1990-04-16 | 1996-12-24 | Credit Managers Association Of California | Method of inactivation of viral and bacterial blood contaminants |
US5556992A (en) * | 1994-09-02 | 1996-09-17 | Universite De Montreal | Novel rhodamine derivatives for photodynamic therapy of cancer and in vitro purging of the leukemias |
-
1998
- 1998-10-09 CN CN98810021A patent/CN1281346A/en active Pending
- 1998-10-09 HU HU0100079A patent/HUP0100079A2/en unknown
- 1998-10-09 IL IL135365A patent/IL135365A0/en unknown
- 1998-10-09 AU AU10722/99A patent/AU754634B2/en not_active Ceased
- 1998-10-09 CA CA002308125A patent/CA2308125A1/en not_active Abandoned
- 1998-10-09 JP JP2000515498A patent/JP2001519368A/en active Pending
- 1998-10-09 NZ NZ503619A patent/NZ503619A/en unknown
- 1998-10-09 BR BR9814816-8A patent/BR9814816A/en not_active Application Discontinuation
- 1998-10-09 EP EP98953317A patent/EP1026988A4/en not_active Withdrawn
- 1998-10-09 KR KR1020007003886A patent/KR20010031056A/en not_active Application Discontinuation
- 1998-10-09 PL PL98341661A patent/PL341661A1/en not_active Application Discontinuation
- 1998-10-09 EA EA200000408A patent/EA200000408A1/en unknown
- 1998-10-09 WO PCT/US1998/021231 patent/WO1999018856A1/en not_active Application Discontinuation
-
2000
- 2000-03-14 NO NO20001322A patent/NO20001322L/en not_active Application Discontinuation
- 2000-03-24 IS IS5414A patent/IS5414A/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5576424A (en) * | 1991-08-23 | 1996-11-19 | Molecular Probes, Inc. | Haloalkyl derivatives of reporter molecules used to analyze metabolic activity in cells |
WO1996033209A1 (en) * | 1995-04-21 | 1996-10-24 | Merck Frosst Canada Inc. | Peptidyl derivatives as inhibitors of pro-apoptotic cysteine proteinases |
WO2000004914A1 (en) * | 1998-07-21 | 2000-02-03 | Cytovia, Inc. | Novel fluorescence dyes and their applications for whole cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof |
Non-Patent Citations (3)
Title |
---|
LENDECKEL U ET AL: "Induction of the membrane alanyl aminopeptidase gene and surface expression in human T-cells by mitogenic activation." THE BIOCHEMICAL JOURNAL. 1 NOV 1996, vol. 319 ( Pt 3), 1 November 1996 (1996-11-01), pages 817-821, XP002301554 ISSN: 0264-6021 * |
See also references of WO9918856A1 * |
TALANIAN R V ET AL: "SUBSTRATE SPECIFICITIES OF CASPASE FAMILY PROTEASES" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 272, no. 15, 11 April 1997 (1997-04-11), pages 9677-9682, XP000652322 ISSN: 0021-9258 * |
Also Published As
Publication number | Publication date |
---|---|
AU754634B2 (en) | 2002-11-21 |
WO1999018856A1 (en) | 1999-04-22 |
EA200000408A1 (en) | 2000-12-25 |
IL135365A0 (en) | 2001-05-20 |
PL341661A1 (en) | 2001-04-23 |
CA2308125A1 (en) | 1999-04-22 |
NO20001322L (en) | 2000-06-13 |
NZ503619A (en) | 2001-11-30 |
BR9814816A (en) | 2004-06-22 |
HUP0100079A2 (en) | 2001-05-28 |
EP1026988A4 (en) | 2005-03-30 |
JP2001519368A (en) | 2001-10-23 |
CN1281346A (en) | 2001-01-24 |
NO20001322D0 (en) | 2000-03-14 |
IS5414A (en) | 2000-03-24 |
KR20010031056A (en) | 2001-04-16 |
AU1072299A (en) | 1999-05-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6342611B1 (en) | Fluorogenic or fluorescent reporter molecules and their applications for whole-cell fluorescence screening assays for capsases and other enzymes and the use thereof | |
AU754634B2 (en) | Novel fluorescent reporter molecules and their applications including assays for caspases | |
US6248904B1 (en) | Fluorescence dyes and their applications for whole-cell fluorescence screening assays for caspases, peptidases, proteases and other enzymes and the use thereof | |
US7879574B2 (en) | Compositions for the detection of enzyme activity in biological samples and methods of use thereof | |
US8623995B2 (en) | Peptide conjugates and fluorescence detection methods for intracellular caspase assay | |
CA2306692C (en) | Dipeptide apoptosis inhibitors and the use thereof | |
US20100068150A1 (en) | Selective Caspase Inhibitors | |
EP2857830B1 (en) | Fluorescent probe for high-sensitivity pancreatic fluid detection, and method for detecting pancreatic fluid | |
Abuelyaman et al. | Fluorescent derivatives of diphenyl [1-(N-peptidylamino) alkyl] phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases | |
AU2008221036B2 (en) | Imaging probes | |
US7312302B2 (en) | Compositions for the detection of enzyme activity in biological samples and methods of use thereof | |
WO2003099780A2 (en) | Luminogenic protease substrates | |
Krafft et al. | [6] Synthetic approaches to continuous assays of retroviral proteases | |
US8697376B2 (en) | Synthetic protease substrates, assay methods using such substrates and kits for practicing the assay | |
MXPA00003443A (en) | Novel fluorescent reporter molecules and their applications including assays for caspases | |
CZ20001158A3 (en) | Novel fluorescent reporting molecules and their use including tests for caspases | |
Makowski et al. | Synthesis of Tetrapeptide p‐nitrophenylanilides containing dehydroalanine and dehydrophenylalanine and their influence on cathepsin C activity | |
Ravula | Synthesis and fluorescence properties of a novel legumain substrate probe | |
Chakrabarty | Development of photo-activatable protease inhibitors and activity-based probes and their application in the study of dynamic proteolytic processes | |
Bogyo | Peptide vinyl sulfones: inhibitors and active site probes for the study of proteasome function in vivo |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20000508 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL PAYMENT 20000508;LT PAYMENT 20000508;LV PAYMENT 20000508;MK PAYMENT 20000508;RO PAYMENT 20000508;SI PAYMENT 20000508 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: ZHANG, HAN-ZHONG Inventor name: DREWE, JOHN, A. Inventor name: KEANA, JOHN, F., W. Inventor name: CAI, SUI, XIONG Inventor name: WEBER, ECKARD |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: 7C 12Q 1/37 B Ipc: 7G 01N 33/58 B Ipc: 7C 12P 17/06 B Ipc: 7G 01N 33/574 B Ipc: 7G 01N 33/48 B Ipc: 7C 12Q 1/70 B Ipc: 7C 12Q 1/00 B Ipc: 7A 61B 8/00 A |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20050215 |
|
17Q | First examination report despatched |
Effective date: 20070320 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: ZHANG, HAN-ZHONG Inventor name: DREWE, JOHN, A. Inventor name: KEANA, JOHN, F., W. Inventor name: CAI, SUI, XIONG Inventor name: WEBER, ECKARD |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
RTI1 | Title (correction) |
Free format text: FLUORESCENT REPORTER MOLECULES AND THEIR APPLICATIONS INCLUDING ASSAYS FOR CASPASES |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20081029 |