Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-11-03
2002-09-24
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S025000, C435S004000, C435S975000
Reexamination Certificate
active
06455268
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the fields of biochemistry and cellular biology. More particularly, the invention relates to compounds and methods for detecting and assaying enzyme activity in an intact cellular system.
BACKGROUND OF THE INVENTION
The detection of hydrolases and oxidases within living cells has been a difficult task. The primary problems associated with these assays are the difficulty in loading cells with the hydrolytic substrate under physiological conditions, leakage of the substrate and product out of the cells, and in the case of fluorescent substrates, high levels of background fluorescence caused by cellular components. Another factor that hinders in vivo determination of hydrolase activity is the pH optimum for fluorescence of the products as compared to the physiological pH. In the case of glycosidases (see e.g. Haugland et al., U.S. Pat. No. 5,208,148; S. N. Fiering et al.,
Cytometry
(1991) 12(4):291-301; M. Lorincz et al.,
Cytometry
(1996) 24(4):321-29; M. Lorincz et al.,
J Biol Chem
(1999) 274(2):657-65; G. P. Nolan et al.,
Proc Natl Acad Sci USA
(1988) 85(8):2603-07; G. D. Yancopoulos et al.,
Mol Cell Biol
(1990) 10(4):1697-704), proteases (D. Irving,
Am Clin Lab
(1997) 16(10):16-17; S. Klingel et al.,
Meth Cell Biol
(1994) 41:449-59; G. Rothe et al.,
Biol Chem Hoppe Seyler
(1992) 373(7):547-54; B. Ulbricht et al.,
Biol Chem Hoppe Seyler
(1995) 376(7):407-14; I. Assfalg-Machleidt et al.,
Biol Chem Hoppe Seyler
(1992) 373(7):433-40; S. Ganesh et al.,
Cytometry
(1995) 20(4):334-40), lipases or esterases, phospholipases A1 and A2 (A2 (T. Meshulam et al.,
J Biol Chem
(1992) 267(30):21465-70), or dealkylases (A. D. Miller et al.,
Meth Cell Biol
(1990) 33:71-79; K. A. Black et al.,
Cytometry
(1993) 14(3):334-38; M. D. Burke et al.,
Biochem J
(1983) 212(1):15-24; A. G. Miller,
Anal Biochem
(1983) 133(1):46-57; H. L. Gurtoo et al.,
Biochem Pharmacol
(1978) 27(22):2659-62), or oxidases and peroxidases (P. Ubezio et al.,
Free Radic Biol Med
(1994) 16(4):509-16), assays in living cells have been developed, but most suffer due to the issues outlined above. In the case of phosphatases (
Proc Natl Acad Sci USA
(1963) 50:1), phospholipases C and D (O. H. Griffith et al.,
Meth Enzymol
(1991) 197:493-502; K. M. Ella et al.,
Anal Biochem
(1994) 218(1):136-42), or sulfatases (C. Stein et al.,
J Biol Chem
(1989) 264(2):1252-59), assays in living cells have not been reported. An assay that overcomes some of the issues for beta-lactamase has been described (G. Zlokarnik et al.,
Science
(1998) 279(5347):84-88; M. Whitney et al.,
Nat Biotechnol
(1998) 16(13):1329-33; and Tsien et al., WO96/30540).
SUMMARY OF THE INVENTION
We have now invented compounds capable of detecting the activity of hydrolytic enzymes intracellularly, in intact cells. In general, the compounds comprise two moieties that are coupled together by a linker that spaces the moieties apart at a distance consistent with fluorescent resonance energy transfer (FRET). The first moiety is selected to be fluorescent, with an emission spectrum that overlaps the excitation spectrum of the second moiety, such that the first moiety is capable of acting as a resonance energy donor. The second moiety is selected to serve as a substrate for the enzyme to be examined, to exhibit fluorescence only after it has been acted upon by the enzyme, and to fluoresce by FRET when stimulated by resonance with the first moiety. The compounds of the invention are hydrophilic, and are retained within the cell after cleavage. The donor moiety fluorescence serves as an internal standard, while the ratio of acceptor moiety fluorescence to donor fluorescence indicates the percentage of compound hydrolyzed by the enzyme in question.
One aspect of the invention is a compound useful for detecting the activity of an enzyme in an intracellular environment, having the structure D—L—A*, where L is a linking molecule that maintains A and D at a distance suitable for FRET, D is a fluorescent donor moiety capable of fluorescing regardless of the presence or absence of the enzyme, and A* is a pre-fluorescent acceptor moiety capable of generating a signal by FRET only after modification to A by the target enzyme (for example, by cleaving a group that prevents fluorescence, or by adding a group that confers fluorescence).
One aspect of the invention is a compound of formula 1:
wherein R
1
is hydroxy, lower alkoxy, lower alkylamino, or di(lower alkyl)amino, R
2
is H or halo, R
3
and R
4
are each independently a sulfate, alkyl, acyl, acyloxymethyl, acyloxyethyl, amino acid, oligopeptide, polypeptide, nucleotide, oligonucleotide, polynucleotide, carbohydrate, polysaccharide, lipid, phospholipid, or a group hydrolysable by a phospholipase, X
1
is OH and X
2
is COOH, or X
1
and X
2
together form —COO—, X
3
is a linking group, and X
4
and X
5
are each independently —O—, —NH—, —LO—, or —LNH—, where L is a cleavable linker.
Another aspect of the invention is a method for measuring enzyme activity within a cell, comprising introducing a compound of the invention into a test cell, permitting an endogenous enzyme (if present) to cleave said substrate into first and second moieties, and measuring the fluorescence. Preferably, the ratio of fluorescence of said first moiety and said second moiety is measured.
Another aspect of the invention is a method of determining gene expression in a host cell, by inserting a polynucleotide encoding a selected enzyme so that it is operatively associated with a native regulatory sequence of said host cell, subjected the host cell to test conditions, contacting the host cell with a compound of the invention that is activated by said selected enzyme, and measuring the signal and/or the change in signal from said compound.
Another aspect of the invention is a method for selecting a compound of the invention for use with a selected enzyme, by providing a panel of host cells, wherein at least one of said host cells expresses a selected enzyme and at least one host cell does not express said enzyme, contacting the panel with at least one candidate compound, and selecting those compounds which provide a FRET signal in host cells which express the selected enzyme.
REFERENCES:
patent: 5208148 (1993-05-01), Haugland et al.
patent: WO 96/30540 (1996-10-01), None
Takakusa et al; Analytical Chemistry, vol. 73(5), p 939-942, 2001.*
Assfalg-Machleidt et al., “Membrane Permeable Fluorogenic Rhodamine Substrates for Selective Determination of Cathepsin L,”0Biol. Chem. Hoppe Seyler373(7):433-440 (1992).
Black et al., “Flow Cytometric Analysis of Xenobiotic Metabolism Activity in Isolated Rat Hepatocytes,”Cytometry14(3):334-338 (1993).
Burke et al., “Fluorescence-Microscopic Measurement of Intracellular Cytochrome P-450 Enzyme Activity (Ethoxyresorufin O-de-Ethylation) in Unfixed Liver Sections,”Biochem. J.212(1):15-24 (1983).
Ella et al., “A Flourescent Assay for Agonist-Activated Phospholipase D in Mammalian Cell Extracts,”Analytical Biochemistry218(1):136-142 (1994).
Fiering, S.N. et al., “Improved FACS-Gal: Flow Cytometric Analysis and Sorting of Viable Eurkaryotic Cells Expressing Reporter Gene Constructs,”Cytometry12(4):291-301 (1991).
Ganesh et al., “Flow Cytometric Determination of Aminopeptidase Activities in Viable Cells Using Fluorogenic Rhodamine 110 Substrates,”Cytometry20(4):334-340 (1995).
Griffith et al., “Phosphatidylinostol Specific: Phospholipases C fromBacillus cereusandBacillus thuringiensis,” Meth. Enzymol.197:493-502 (1991).
Gurtoo et al., “Cytochrome P-450 in a Cultured Human Lymphocyte Cell Line,”Biochemical Pharmacology27(22):2659-2662 (1978).
Klingel et al., “Flow Cytometric Determination of Cysteine and Serine Proteinase Activities in Living Cells with Rhodamine 110 Substrates,”Methods in Cell Biol.41:449-459 (1994).
Lorincz et al., “Enzyme-Generated Intracellular Fluorescence for Single-Cell Reporter Gene Analysis UtilizingEscherichia Coli&bgr;-Glucuronidase,”Cytometry24(4):321-329 (1996).
Lorincz et al., “Single Cell Analysis and
Jarnigan Kurt
Lee Ving J.
Leger Roger
Morgans David
Iconix Pharmaceuticals Inc.
Leary Louise N.
Robins & Pasternak LLP
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