EP1759015A2 - Fluorogene enzymassays und substrate für kinasen und phosphatasen - Google Patents
Fluorogene enzymassays und substrate für kinasen und phosphatasenInfo
- Publication number
- EP1759015A2 EP1759015A2 EP05762562A EP05762562A EP1759015A2 EP 1759015 A2 EP1759015 A2 EP 1759015A2 EP 05762562 A EP05762562 A EP 05762562A EP 05762562 A EP05762562 A EP 05762562A EP 1759015 A2 EP1759015 A2 EP 1759015A2
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- EP
- European Patent Office
- Prior art keywords
- moiety
- charge
- substrate
- molecule
- balance
- 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.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
Definitions
- the present disclosure relates to compositions, methods and kits for detecting, quantifying and/or characterizing enzymes in a sample.
- Enzymes are molecules that increase the rate of chemical reactions. Enzymatic assays for detecting, quantifying and/or characterizing enzyme activity have significant biological, medical and industrial applications, hi biological systems, enzymes are involved in synthesis and replication of nucleic acids, modification, and degradation of polypeptides, synthesis of metabolites, and many other functions. In medical testing, enzymes are important indicators of the health or disease of human patients. In industry, enzymes are used for many purposes, such as proteases used in laundry detergents, metabolic enzymes to make specialty chemicals such as amino acids and vitamins, and chirally specific enzymes to prepare enantiomerically pure drugs. Assays using reporter molecules are important tools for studying and detecting enzymes that mediate numerous biological and industrial processes. Although numerous approaches have been developed for assaying enzymes using reporter molecules, there remains a need to find new assay designs that can be used to inexpensively and conveniently detect and characterize a wide variety of enzymes.
- compositions, methods and kits useful for, among other things, detecting, quantifying and/or characterizing enzymes.
- the compositions generally comprise one or more molecules that collectivity include three to four different types of moieties: a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety.
- the hydrophobic moiety acts to integrate the molecule(s) of the composition into a micelle when included in an aqueous solvent at or above its critical micelle concentration (CMC).
- CMC critical micelle concentration
- the fluorescent moiety functions to produce a fluorescent signal when the substrate moiety of the composition is acted upon by an enzyme.
- the substrate moiety comprises a recognition moiety comprising a substrate or putative substrate for an enzyme of interest.
- the charge- balance moiety acts to balance the overall charge of the composition. While not intending to be bound by any theory of operation, it is believed that balancing the overall net charge acts to promote or encourage micelle formation.
- a mixture comprising a sample and one or more protein kinase recognition moieties.
- a recognition moiety comprise all or a part of a consensus sequence for a protein kinase.
- the consensus sequence includes at least one unphosphorylated residue that is capable of being phosphorylated by a protein kinase.
- each independently of the other can comprise all, or part of a consensus sequence for a protein kinase.
- the consensus sequences are selected such that they, either individually or together, provide two or more unphosphorylated residues that are capable of being phosphorylated by a protein kinase.
- the unphosphorylated residue(s) in the consensus sequence may be any residue that includes a group that is capable of being phosphorylated by a protein kinase.
- the residue is a tyrosine residue.
- the residue is a serine residue.
- the residue is a threonine residue.
- the consensus sequence can comprise more than one residue capable of being phosphorylated.
- the residues may be the same, some of them may be the same and others different, or they may all differ from one another.
- the recognition moieties may be the same, or they may all differ from one another.
- the protein kinases to be detected can be any protein kinase known in the art.
- the protein kinase comprises protein kinase A.
- the protein kinase comprises protein kinase C.
- the protein kinase comprises a protein kinase candidate, and a method is used to confirm and/or characterize the kinase activity of the candidate.
- the protein kinase consensus sequence can be designed to be reactive with a particular protein kinase or a group of protein kinases, or it can be designed to determine substrate specificity and/or other catalytic features, such as determining a value for kcat or Km.
- the recognition moiety may include additional amino acid residues (or analogs thereof) that facilitate binding specificity, affinity, and/or rate of phosphorylation by the protein kinase(s) to be detected.
- the recognition moiety comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. In other embodiments, the recognition moiety can comprise more than 20 amino acid residues.
- the disclosure provides methods for detecting the phosphatase activity of one or more protein phosphatases in a sample.
- a mixture comprising a sample and at least one phosphatase recognition moiety, wherein the phosphatase recognition moeity comprises (a) a consensus sequence comprising at least one phosphorylated residue that is capable of being dephosphorylated (hydrolyzed) by a phosphatase, (b) one or more hydrophobic moieties, and (c) at least one fluorescent moiety(ies).
- the mixture is subjected to conditions effective to allow dephosphorylation of the phosphorylated residues when a phosphatase is present in the sample, thereby increasing a fluorescent signal produced by the fluorescent moiety. Detection of an increase in a fluorescent signal indicates the presence of a phosphatase in the sample.
- each, independently of the other can comprise all, or part of the consensus sequence for a phosphatase.
- the phosphatase consensus sequences are selected such that they, either individually or together, provide two or more phosphorylated residues that are capable of being dephosphorylated by a phosphatase.
- the phosphorylated residue(s) in the phosphatase consensus sequence may be any residue that includes a group that is capable of being dephosphorylated by a phosphatase.
- the residue is a phosphotyrosine residue.
- the residue is a phosphoserine residue.
- the residue is a phosphothreonine residue.
- the residues may be the same, some of them may be the same and others different, or they may all differ from one another. Additionally, the recognition moieties may be the same, or they may all differ from one another.
- the phosphatase to be detected can be any phosphatase known in the art.
- the phosphatase can be a phosphatase candidate, and the method used to confirm and/or characterize the phosphatase activity of the candidate.
- the phosphatase consensus sequence can be designed to be reactive with a particular phosphatase or a group of phosphatases, or it can be designed to determine substrate specificity and other catalytic features, such as determining a value for kcat or Km.
- the recognition moiety may include additional amino acid residues (or analogs thereof) that facilitate binding specificity, affinity, and/or rate of dephosphorylation by the phosphatase(s).
- the recognition moiety comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues.
- the hydrophobic moieties are selected such that they, either individually or together, are capable of integrating the substrate molecule and the charge-balance molecule into a micelle.
- the hydrophobic moiety(ies) comprise a saturated or unsaturated hydrocarbon comprising from 6 to 30 carbon atoms.
- the hydrophobic moiety(ies) comprise a hydrocarbon chain corresponding in structure to a hydrocarbon chain or "tail" of a naturally occurring fatty acid, lipid or phospholipid.
- the hydrophobic moiety(ies) facilitate an increase in fluorescence of the fluorescent moiety upon phosphorylation of the substrate that is greater than the amplitude of the increase that would be obtained with the same substrate structure lacking a hydrophobic moiety.
- hydrophobic moieties comprising the various molecules can be the same, some of them can be the same and others different, or they may all differ from one another.
- the hydrophobic moieties comprising the substrate molecule and the charge-balance molecule can be the same. In other embodiments, the hydrophobic moieties comprising the substrate molecule and the charge-balance molecule can differ from each other.
- the hydrophobic, fluorescent, substrate, and charge-balance moieties can be included in a single molecule, or they can be included in different molecules.
- the composition comprises a substrate molecule that comprises a hydrophobic moiety capable of integrating the substrate molecule into a micelle, a fluorescent moiety, a substrate moiety, and a charge-balance moiety.
- the composition comprises two distinct molecules, a substrate molecule and a charge-balance molecule.
- the substrate molecule comprises a hydrophobic moiety and a substrate moiety.
- the charge-balance molecule comprises a hydrophobic moiety and a charge-balance moiety.
- One or both of the substrate and/or charge-balance molecules further comprises a fluorescent moiety.
- suitable fluorescent dyes include xanthene dyes such as fluorescein, sulfofluorescein and rhodamine dyes, cyanine dyes, bodipy dyes and squaraine dyes. Fluorescent moieties comprising other fluorescent dyes may also be used.
- the various substrate and/or charge-balance molecules can comprise additional moieties.
- a substrate molecule can comprise a charge-balance moiety and vice-versa.
- the compositions can comprise a quenching moiety.
- the quenching moiety can be included in the substrate molecule, the charge-balance molecule, in both the substrate molecule and charge-balance molecule, or in a distinct quenching molecule.
- a quenching molecule comprises a hydrophobic moiety and a quenching moiety.
- the quenching moiety can be any moiety capable of quenching the fluorescence of a fluorescent moiety when the quenching moiety is in close proximity to the fluorescent moiety.
- moieties described herein i.e., substrate moiety(ies), fluorescent moiety(ies), the hydrophobic moiety(ies), and the charge balance moiety(ies) can be connected in any way that permits them to perform their respective functions.
- one or more of the moieties can be linked directly to each other.
- one or more of the moieties can be linked indirectly to each other through a linker.
- the present disclosure provides methods for detecting or measuring an enzyme activity.
- a mixture comprising a sample and one or ore molecules that collectively include three or four types of moieties: (a) substrate moiety comprisiing a recognition moiety for an enzyme, (b) a hydrophobic moeity, (c) a fluorescent moiety, and (d) a charge-balance moiety.
- the mixture is subjected to conditions effective to allow the enzyme to modify the recognition moiety to produce a fluorescently detectable product.
- the enzyme is a protein kinase. In other embodiments, the enzyme is a protein phosphatase.
- FIG. 1 illustrates an exemplary embodiment of an enzyme assay scheme utilizing an exemplary embodiment of a single molecule comprising a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety.
- FIG. 2 illustrates an exemplary embodiment of an enzyme assay scheme utilizing an exemplary embodiment of a substrate molecule and a charge-balance molecule.
- FIG. 3 illustrates an exemplary embodiment of an enzyme assay scheme utilizing an exemplary embodiment of a substrate molecule, charge-balance molecule and a quenching molecule.
- FIGS. 4A-I illustrate exemplary embodiments of substrate molecules comprising one or more hydrophobic moiety(ies), fluorescent moiety(ies), and a substrate moiety comprising two or more recognition moieties.
- FIG 5 illustrates an exemplary embodiment of a substrate molecule comprising a substrate moiety comprising two recognition moieties, one hydrophobic moiety and a fluorescent moiety.
- FIG. 6 illustrates an exemplary embodiment of a substrate molecule comprising a substrate moieties, comprising two recognition moieties, two hydrophobic moieties and a fluorescent moiety.
- FIGS. 7A-D illustrate exemplary embodiments of substrate molecules comprising a hydrophobic moiety, a charge-balance moiety(ies), a fluorescent moiety, and a substrate moiety.
- FIGS. 8A-H illustrate exemplary embodiments of substrate molecules (FIGS. 8 A, C, E, G) and charge-balance molecules (FIGS. 8B, D, F, H).
- FIGS. 9A-B illustrate exemplary embodiments of a substrate molecule (FIG. 9A) and a charge-balance molecule (FIG. 9B).
- FIG. 1OA shows the rate of reaction for a kinase substrate, i.e., CiiOOK(Dye2)RRIPLSPLSPOOK(C ⁇ )-NH 2 (8 ⁇ M) for 10 and 100 ⁇ M ATP.
- a kinase substrate i.e., CiiOOK(Dye2)RRIPLSPLSPOOK(C ⁇ )-NH 2 (8 ⁇ M) for 10 and 100 ⁇ M ATP.
- FIG. 1OB shows the rate of reaction for a kinase substrate, i.e., C n OOK(Dye2)RRIPLSPOOK(Ci i)-NH 2 (8 ⁇ M) for 10 and 100 ⁇ M ATP.
- FIG. 11 shows the addition of varying concentrations (0, 5, 10, 20, 50 ⁇ M) of a ' charge-balance molecule, C i 6 RROOORRIYGRF quenching the fluorescence of a substrate molecule, C 16 K(Dye2)OOOEEIYGEF (10 ⁇ M) in 25 niM Tris (pH 7.6).
- FIG. 12 shows the rate of reaction of 5 nM tyrosine kinase (Lyn) against the substrate molecule C 16 K(Dye2)OOOEEIYGEF (2 ⁇ M), charge-balance molecule C 16 RROOORRTYGRF (2 ⁇ M), with 0 and 100 ⁇ M ATP.
- Detect and “detection” have their standard meaning, and are intended to encompass detection, measurement, and characterization of a selected enzyme or enzyme activity.
- enzyme activity can be “detected” in the course of detecting, screening for, or characterizing inhibitors, activators, and modulators of the enzyme activity.
- Fatty Acid has its standard meaning and is intended to refer to a long-chain hydrocarbon carboxylic acid in which the hydrocarbon chain is saturated, mono-unsaturated or polyunsaturated.
- the hydrocarbon chain can be linear, branched or cyclic, or can comprise a combination of these features, and can be unsubstituted or substituted.
- Fatty acids typically have the structural formula RC(O)OH, where R is a substituted or unsubstituted, saturated, mono-unsaturated or polyunsaturated hydrocarbon comprising from 6 to 30 carbon atoms which has a structure that is linear, branched, cyclic or a combination thereof.
- Micelle has its standard meaning and is intended to refer to an aggregate formed by amphipathic molecules in water or an aqueous environment such that their polar ends or portions are in contact with the water or aqueous environment and their nonpolar ends or portions are in the interior of the aggregate.
- a micelle can take any shape or form, including but not limited to, a non- lamellar "detergent-like” aggregate that does not enclose a portion of the water or aqueous environment, or a unilamellar or multilamellar "vesicle-like” aggregate that encloses a portion of the water or aqueous environment, such as, for example, a liposome.
- Quench has its standard meaning and is intended to refer to a reduction in the fluorescence intensity of a fluorescent group or moiety as measured at a specified wavelength, regardless of the mechanism by which the reduction is achieved.
- the quenching can be due to molecular collision, energy transfer such as FRET, photoinduced electron transfer such as PET, a change in the fluorescence spectrum (color) of the fluorescent group or moiety or any other mechanism (or combination of mechanisms).
- the amount of the reduction is not critical and can vary over a broad range. The only requirement is that the reduction be detectable by the detection system being used. Thus, a fluorescence signal is "quenched” if its intensity at a specified wavelength is reduced by any measurable amount.
- a fluorescence signal is "substantially quenched” if its intensity at a specified wavelength is reduced by at least 50%, for example by 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100%.
- Polypeptide sequences are provided with an orientation (left to right) of the N terminus to C terminus, with amino acid residues represented by the standard 3-letter or 1- letter codes (e.g., Stryer, L., Biochemistry, 2 nd Ed., W.H. Freeman and Co., San Francisco, CA, page 16 (1981)).
- compositions, methods and kits useful for, among other things, detecting, quantifying and/or characterizing enzymes.
- the compositions typically form micelles comprising one or more molecules that collectively include a number of different moieties, such as a hydrophobic moiety, a fluorescent moiety, a substrate moiety, and a charge-balance moiety.
- the hydrophobic moieties are capable of anchoring or integrating the molecules into the micelle.
- the exact numbers, lengths, sizes and/or composition of the hydrophobic moieties can be varied.
- each hydrophobic moiety may be the same, or some or all of the hydrophobic moieties may differ.
- the substrate moiety comprises at least one recognition moiety comprising a substrate or a putative substrate that can be acted upon by a specific enzyme or agent.
- the fluorescence signal of the fluorescent moiety is quenched when the substrate moiety and/or the charge-balance moiety is integrated into the micelle.
- the recognition moiety When the recognition moiety is acted upon by the specified enzyme it promotes the dissociation of the fluorescent moiety from the micelle, thereby reducing or eliminating the quenching effect caused by the interactions between the fluorescent moiety and the micelle.
- the dissociation may be caused by cleavage of the substrate or by the addition, deletion, or substitution of chemical groups, such as phosphate groups, which can destabilize the substrate moiety in the micelle, promoting its release therefrom.
- the substrate moieties described herein can be used in a continuous monitoring phase, in real time, to allow the user to rapidly determine whether enzyme activity is present in the sample, and optionally, the amount or specific activity of the enzyme.
- the charge-balance moiety acts to balance the overall charge of the micelle. For example, if the substrate moiety comprises one or more charged chemical groups, the presence of these groups can destabilize the substrate moiety in the micelle, thereby promoting the release of the substrate moiety from the micelle in the absence of the specified enzyme.
- Release of the charged substrate moiety from the micelle can be prevented by including a charge-balance moiety designed to counter the charge of the substrate moeity via the inclusion of chemical groups that have the opposite charge of the chemical groups comprising the substrate moiety, such that the overall charge of the micelle is neutral.
- a charge-balance moiety designed to counter the charge of the substrate moeity via the inclusion of chemical groups that have the opposite charge of the chemical groups comprising the substrate moiety, such that the overall charge of the micelle is neutral.
- the charge-balance moiety stable micelles can be formed in the presence of destabilizing chemical groups.
- the substrate moiety When the substrate moiety is acted upon by the specified enzyme it promotes destabilization of the micelle, for example, by the addition of charged groups, and dissociation of the fluorescent moiety from the micelle, thereby reducing or eliminating the quenching effect and producing a detectable increase in fluorescence.
- the micelle comprises a single molecule that includes a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety.
- the micelle comprises two distinct molecules, a substrate molecule and a charge-balance molecule.
- the substrate molecule comprises a hydrophobic moiety and a substrate moiety.
- the charge-balance molecule comprises a hydrophobic moiety and a charge-balance moiety.
- the substrate moiety can comprise one or more enzyme recognition moieties.
- the enzyme recognition moieties can comprise all, or part of, a consensus sequence for a given enzyme, for example, a protein kinase enzyme.
- One or both of the substrate molecule and/or charge-balance molecule further comprises a fluorescent moiety.
- the moieties can be connected to each other in any way that permits them to perform their respective functions.
- the micelle can comprise additional molecules such as a quenching molecule.
- the quenching molecule can include a hydrophobic moiety and a quenching moiety that quenches the fluorescence of the fluorescent moiety.
- the quenching moiety can be positioned so that it is able to intramolecularly quench the fluorescence of the fluorescent moiety on the substrate molecule and/or the charge-balance molecule, which includes it, or, alternatively, the quenching moiety may be positioned so that intramolecular quenching does not occur.
- the quenching moiety may intermolecularly quench the fluorescence of a fluorescent moiety on another molecule in the micelle which is in close proximity thereto.
- the hydrophobic moiety(ies) act to anchor or integrate the various molecules described herein into the micelle.
- the exact numbers, lengths, size and/or compositions of the hydrophobic moieties can be varied.
- each hydrophobic moiety may be the same, or some or all of the hydrophobic moieties may differ.
- the composition comprises two distinct molecules, a substrate molecule and a charge-balance molecule, each which can comprise a hydrophobic moiety.
- the hydrophobic moiety(ies) of the substrate molecule can be the same length, size and/or composition from the hydrophobic moiety(ies) of the charge-balance molecule.
- the hydrophobic moiety(ies) of the substrate molecule can differ in length, size and/or composition from the hydrophobic moiety(ies) of the charge-balance molecule.
- the hydrophobic moieties comprise a substituted or unsubstituted hydrocarbon of sufficient hydrophobic character (e.g., length and/or size) to cause the substrate molecule and/or the charge-balance molecule to become integrated or incorporated into a micelle when the molecule(s) is placed in an aqueous environment at a concentration above a micelle-forming threshold, such as at or above its critical micelle concentration (CMC).
- CMC critical micelle concentration
- the hydrophobic moieties comprise a substituted or unsubstituted hydrocarbon comprising from 6 to 30 carbon atoms, or from 6 to 25 carbon atoms, or from 6 to 20 carbon atoms, or from 6 to 15 carbon atoms, or from 8 to 30 carbon atoms, or from 8 to 25 carbon atoms, or from 8 to 20 carbon atoms, or from 8 to 15 carbon atoms, or from 12 to 30 carbon atoms, or from 12 to 25 carbon atoms, or from 12 to 20 carbon atoms.
- the hydrocarbon can be linear, branched, cyclic, or any combination thereof, and can optionally include one or more of the same or different substituents.
- Exemplary linear hydrocarbon groups comprise C6, C7, C8, C9, ClO, Cl 1, C12, C13, C14, C 15, C 16, C 17, C 18, C 19, C20, C22, C24, and C26 alkyl chains.
- the hydrophobic moieties are fully saturated.
- the hydrophobic moieties can comprise one or more carbon-carbon double bonds which can be, independently of one another, in the cis or trans configuration, and/or one or more carbon-carbon triple bonds.
- the hydrophobic moieties can have one or more cycloalkyl groups, or one or more aryl rings or arylalkyl groups, such as one or two phenyl rings.
- the hydrophobic moiety is a nonaromatic moiety that does not have a cyclic aromatic pi electron system. In some embodiments, if the hydrophobic moiety contains one or more unsaturated carbon-carbon bonds, those carbon-carbon bonds are not conjugated. In another embodiment, the structure of the hydrophobic moiety is incapable of interacting with the fluorescent moiety, by a FRET or stacking interaction, to quench fluorescence of the fluorescent moiety. Also encompassed herein are embodiments that involve a combination of any two or more of the foregoing embodiments. Optimization testing can be done by making several substrate and/or charge-balance molecules having different hydrophobic moieties.
- the molecule(s) of the composition comprises two hydrophobic moieties linked to the Cl and C2 carbons of a glycerolyl group via ester linkages (or other linkages).
- the two hydrophobic moieties can be the same or they can differ from another.
- each hydrophobic moiety is selected to correspond to the hydrocarbon chain or "tail" of a naturally occurring fatty acid
- the hydrophobic moieties are selected to correspond to the hydrocarbon chains or tails of a naturally occurring phospholipid.
- Non-limiting examples of hydrocarbon chains or tails of commonly occurring fatty acids are provided in Table 1, below:
- the hydrophobic moieties comprise amino acids or amino acid analogs that have hydrophobic side chains.
- the amino acids or analogs are chosen to provide sufficient hydrophobicity to integrate the molecule(s) of the composition into a micelle under the assay conditions used to detect the enzymes.
- Exemplary hydrophobic amino acids include alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine as described in Alberts, B., et ai, Molecular Biology of the Cell, 4 th Ed., Garland Science, New York, NY, Figure 3.3 (2002)).
- amino acid analogs include norvaline, aminobutyric acid, cyclohexylalanine, butylglycine, phenylglycine, and N-methylvaline (see “Amino Acids and Amino Acid Analogs” section 2002-2003 Novabiochem catalog).
- hydrophobicity of a hydrophobic moiety can be calculated by assigning each amino acid a hydrophobicity value and then averaging the values along the hydrophobic moiety. Hydrophobicity values for the common amino acids are shown Table 2.
- the hydrophobic moiety comprises the same amino acid or amino acid analog.
- the hydrophobic moiety can be poly(leucine), comprising from 1 to 10 leucine residues.
- the hydrophobic moiety comprises a mixture of amino acids or amino acid analogs.
- the hydrophobic moiety can comprise a mixture of amino acids, such as leucine and isoleucine, from 1 to 10 leucine resides and from 1 to 10 isoleucine residues can be used.
- the hydrophobic moiety can comprise a mixture of amino acids, amino acid analogs, and hydrocarbons.
- the hydrophobic moiety can comprise from 1 to 10 residues of the amino acids or amino acid analogs and a hydrocarbon comprising from 2 to 30 carbons atoms.
- the hydrophobic moieties can be connected to the other moieties comprising the substrate molecule and/or the charge-balance molecule in any way that permits them to perform their respective functions.
- the moieties can be connected directly to one another, i.e., covalently linked to each other.
- one, some, or all of the moieties can be connected indirectly to one another, i.e., via one or more optional linkers.
- the hydrophobic moiety is distinct from the fluorescent moiety because the hydrophobic moiety does not comprise any of the atoms in the fluorescent moiety that are part of the aromatic or conjugated pi-electron system that produces the fluorescent signal.
- a hydrophobic moiety is connected to the C4 position of a xanthene ring (e.g., the C4' position of a fluorescein or rhodamine dye)
- the hydrophobic moiety does not comprise any of the aromatic ring atoms of the xanthene ring.
- the substrate molecule and/or the charge-balance molecule can further comprise one or more fluorescent moiety(ies) which can be selectively "turned on” when the substrate molecule and/or micelle is acted upon by an enzyme or agent as described herein.
- the fluorescent moiety can comprise any entity that provides a fluorescent signal and that can be used in accordance with the methods and principles described herein. In the exemplary embodiment illustrated in FIG.l, the fluorescence of the fluorescent moiety is quenched when the substrate molecule is incorporated into the micelle. When the substrate moiety is acted upon by a specified enzyme it results in the dissociation of the substrate molecule and/or micelle resulting in the release of the fluorescent moiety, thereby increasing the fluorescent signal produced by the fluorescent moiety.
- the fluorescent moiety(ies) can be connected to the other moieties comprising the substrate molecule and/or the charge-balance molecule in any way that permits them to perform their respective functions.
- the moieties can be connected directly to one another, i.e., covalently linked to each other.
- one, some or all of the moieties can be connected indirectly to one another, i.e., via one or more optional linkers.
- Quenching of the fluorescent moiety within the micelle can be achieved in a variety of different ways.
- the quenching effect may be achieved or caused by "self-quenching.”
- Self-quenching can occur when the substrate molecule and/or the charge- balance molecule comprising a fluorescent moiety are present in the micelle at a concentration sufficient or molar ratio high enough to bring their fluorescent moieties in close enough proximity to one another such that their fluorescence signals are quenched. Release of the fluorescent moieties from the micelle reduces or abolishes the "self-quenching," producing an increase in their fluorescence signals.
- a fluorescent moiety is "released” or “removed” from a micelle if any molecule or molecular fragment that contains the fluorescent moiety is released or removed from the micelle.
- the fluorescent moiety can be soluble or insoluble.
- the fluorescent moiety is soluble under conditions of the assay so as to facilitate removal of the released fluorescent moiety from the micelle into the assay medium.
- the fluorescent moiety is insoluble under conditions of the assay so that the fluorescent moiety can precipitate out of solution and localize at the site at which it was produced, thereby producing an increase in the fluorescent signal as compared to the signal observed in solution.
- the quenching effect can be achieved or caused by other moieties comprising the micelle. These moieties are referred to as "quenching moieties," regardless of the mechanism by which the quenching is achieved. Such quenching moieties and quenching molecules are described in more detail, below.
- quenching moieties and quenching molecules are described in more detail, below.
- the degree of quenching achieved within the micelle is not critical for success, provided that it is measurable by the detection system being used. As will be appreciated, higher degrees of quenching are desirable, because the greater the quenching effect, the lower the background fluorescence prior to removal of the quenching effect. In theory, a quenching effect of 100%, which corresponds to complete suppression of a measurable fluorescence signal, would be ideal. In practice, any measurable amount will suffice.
- the amount and/or molar percentage of substrate molecule and/or the charge-balance molecule and optional quenching molecule in a micelle necessary to provide a desired degree of quenching in the micelle can vary depending upon, among other factors, the choice of the fluorescent moiety.
- the amount and/or molar percentage of any substrate molecule and/or the charge-balance molecule (or mixture of substrate molecules and/or the charge-balance molecules) and optional quenching molecule (or mixture of optional quenching molecules) comprising a substrate molecule and/or the charge-balance molecule-containing micelle in order to obtain a sufficient degree of quenching can be determined empirically.
- the fluorescent moiety of the substrate molecule and/or the charge-balance molecule comprises a fluorescent dye that in turn comprises a resonance-delocalized system or aromatic ring system that absorbs light at a first wavelength and emits fluorescent light at a second wavelength in response to the absorption event.
- a fluorescent dye molecules are known in the art.
- fluorescent dyes can be selected from any of a variety of classes of fluorescent compounds, such as xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines, squaraines, bodipy dyes, coumarins, oxazines, and carbopyronines.
- the fluorescent moiety comprises a xanthene dye.
- xanthene dyes are characterized by three main features: (1) a parent xanthene ring; (2) an exocyclic hydroxyl or amine substituent; and (3) an exocyclic oxo or imminium substituent.
- the exocyclic substituents are typically positioned at the C3 and C6 carbons of the parent xanthene ring, although "extended" xanthenes in which the parent xanthene ring comprises a benzo group fused to either or both of the C5/C6 and C3/C4 carbons are also known. In these extended xanthenes, the characteristic exocyclic substituents are positioned at the corresponding positions of the extended xanthene ring.
- a "xanthene dye” generally comprises one of the following parent rings:
- a 1 is OH or NH 2 and A 2 is O or NH 2 +.
- a 1 is OH and A 2 is O
- the parent ring is a fluorescein-type xanthene ring.
- a 1 is NH 2 and A 2 is NH 2 +
- the parent ring is a rhodamine-type xanthene ring.
- a 1 is NH 2 and A 2 is O
- the parent ring is a rhodol-type xanthene ring.
- One or both of nitrogens of A 1 and A 2 (when present) and/or one or more of the carbon atoms at positions Cl, C2, C2", C4, C4", C5, C5", C7", C7 and C8 can be independently substituted with a wide variety of the same or different substituents.
- typical substituents comprise, but are not limited to, -X, -R a , -OR a , -SR a , -NR a R a , perhalo (C 1 -C 6 ) alkyl, -CX 3 , -CF 3 , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO 2 , -N 3 , -S(O) 2 O-, -S(O) 2 OH, -S(O) 2 R 3 , -C(O)R, -C(O)X, -C(S)R a , -C(S)X, -C(O)OR a , -C(O)O " , -C(S)OR 3 , -C(O)SR 3 , -C(S)SR 3 , -C(O)NR 3 R 3 , -C(S)
- substituents which do not tend to completely quench the fluorescence of the parent ring are preferred, but in some embodiments quenching substituents may be desirable.
- Substituents that tend to quench fluorescence of parent xanthene rings are electron-withdrawing groups, such as -NO 2 , -Br and -I.
- Cl and C2 substituents and/or the C7 and C8 substituents can be taken together to form substituted or unsubstituted buta[l,3]dieno or (C 5 -C 20 ) aryleno bridges.
- exemplary parent xanthene rings including unsubstituted benzo bridges fused to the C1/C2 and C7/C8 carbons are illustrated below:
- the benzo or aryleno bridges may be substituted at one or more positions with a variety of different substituent groups, such as the substituent groups previously described above for carbons C1-C8 in structures (Ia)-(Ic), supra.
- substituent groups such as the substituent groups previously described above for carbons C1-C8 in structures (Ia)-(Ic), supra.
- the substituents may all be the same, or some or all of the substituents can differ from one another.
- the nitrogen atoms may be included in one or two bridges involving adjacent carbon atom(s).
- the bridging groups may be the same or different, and are typically selected from (C 1 -Ci 2 ) alkyldiyl, (Ci-Ci 2 ) alkyleno, 2-12 membered heteroalkyldiyl and/or 2-12 membered heteroalkyleno bridges.
- Non-limiting exemplary parent rings that comprise bridges involving the exocyclic nitrogens are illustrated below:
- the parent ring may also comprise a substituent at the C9 position.
- the C9 substituent is selected from acetylene, lower (e.g., from 1 to 6 carbon atoms) alkanyl, lower alkenyl, cyano, aryl, phenyl, heteroaryl, electron-rich heteroaryl and substituted forms of any of the preceding groups.
- the parent ring comprises benzo or aryleno bridges fused to the C1/C2 and C7/C8 positions, such as, for example, rings (Id), (Ie) and (If) illustrated above, the C9 carbon is preferably unsubstituted.
- the C9 substituent is a substituted or unsubstituted phenyl ring such that the xanthene dye comprises one of the following structures:
- the carbons at positions 3, 4, 5, 6 and 7 may be substituted with a variety of different substituent groups, such as the substituent groups previously described for carbons C1-C8.
- the carbon at position C3 is substituted with a carboxyl (-COOH) or sulfuric acid (-SO 3 H) group, or an anion thereof.
- Dyes of formulae (Ha), (lib) and (lie) in which A 1 is OH and A 2 is O are referred to herein as fluorescein dyes; dyes of formulae (Ha), (lib) and (lie) in which A 1 is NH 2 and A 2 is NH 2 + are referred to herein as rhodamine dyes; and dyes of formulae (Ha), (lib) and (He) in which A 1 is OH and A 2 is NH 2 + (or in which A 1 is NH 2 and A 2 is O) are referred to herein as rhodol dyes.
- carboxy rhodamine and fluorescein dyes may exist in a lactone form.
- the fluorescent moiety comprises a rhodamine dye.
- exemplary suitable rhodamine dyes include, but are not limited to, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX), 4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G), 4,7-dichlororhodamine 6G, rhodamine 110 (Rl 10), 4,7-dichlororhodamine 110 (dRl 10), tetramethyl rhodamine (TAMRA) and 4,7-dichloro-tetramethylrhodamine (dT AMRA).
- rhodamine dyes include, for example, those described in U.S. Patents Nos. 6,248,884, 6,111,116, 6,080,852, 6,051,719, 6,025,505, 6,017,712, 5,936,087, 5,847,162, 5,840,999, 5,750,409, 5,366,860, 5,231,191, and 5,227,487; PCT Publications WO 97/36960 and WO 99/27020; Lee et al, NuCL. ACIDS RES.
- rhodamine dyes are 4,7,- dichlororhodamines.
- the fluorescent moiety comprises a 4,7-dichloro- orthocarboxyrhodamine dye.
- the fluorescent moiety comprises a fluorescein dye.
- fluorescein dyes described in U.S. Patents 6,008,379, 5,840,999, 5,750,409, 5,654,442, 5,188,934, 5,066,580, 4,933,471, 4,481,136 and 4,439,356; PCT Publication WO 99/16832, and EPO Publication 050684.
- a preferred subset of fluorescein dyes are 4,7-dichlorofluoresceins.
- Other preferred fluorescein dyes include, but are not limited to, 5-carboxyfluorescein (5-FAM) and 6-carboxyfluorescein (6-FAM).
- the fluorescein moiety comprises a 4,7 -dichloro- orthocarboxyfiuorescein dye.
- the fluorescent moiety can include a cyanine, a phthalocyanine, a squaraine, or a bodipy dye, such as those described in the following references and the references cited therein: U.S. Patent Nos. 6,080,868, 6,005,113, 5,945,526, 5,863,753, 5,863,727, 5,800,996, and 5,436,134; and PCT Publication WO 96/04405.
- the fluorescent moiety can comprise a network of dyes that operate cooperatively with one another such as, for example by FRET or another mechanism, to provide large Stoke's shifts.
- Such dye networks typically comprise a fluorescence donor moiety and a fluorescence acceptor moiety, and may comprise additional moieties that act as both fluorescence acceptors and donors.
- the fluorescence donor and acceptor moieties can comprise any of the previously described dyes, provided that dyes are selected that can act cooperatively with one another.
- the fluorescent moiety comprises a fluorescence donor moiety which comprises a fluorescein dye and a fluorescence acceptor moiety which comprises a fluorescein or rhodamine dye.
- suitable dye pairs or networks are described in U.S. Patent Nos. 6,399,392, 6,232,075, 5,863,727, and 5,800,996.
- a substrate moiety comprises a substrate or putative substrate that can be acted upon by specified enzymes or agents. Any type of enzyme or chemical reaction on the substrate moiety/micelle can be used, provided that it is capable of producing a detectable change (e.g., an increase) in fluorescence.
- the specified enzyme is substantially active at the interface between the micelle and the assay medium. Selection of a particular enzyme or chemical reaction may depend, in part, on the structure of the substrate moiety, as well as on other factors.
- the enzyme or agent acts upon the substrate moiety to cleave the substrate moiety.
- the substrate moiety comprises a cleavage site that is cleavable by a chemical reagent or cleaving enzyme.
- the substrate moiety can comprise a cleavage site that is cleavable by a lipase, a phospholipase, a peptidase, a nuclease or a glycosidase enzyme.
- the substrate moiety may further comprise additional residues and/or features that facilitate the specificity, affinity and/or kinetics of the cleaving enzyme.
- cleaving enzyme "motifs" can comprise the cleavage site or, alternatively, the cleavage site may be external to the motif.
- motifs can comprise the cleavage site or, alternatively, the cleavage site may be external to the motif.
- certain endonucleases cleave at positions that are upstream or downstream of the region of the nucleic acid molecule bound by the endonuclease.
- the chemical composition of the substrate moiety will depend upon, among other factors, the requirements of the cleaving enzyme.
- the cleaving enzyme is a protease
- the substrate moiety can comprise a peptide (or analog thereof) recognized and cleaved by the particular protease.
- the cleaving enzyme is a nuclease
- the substrate moiety can comprise an oligonucleotide (or analog thereof) recognized and cleaved by a particular nuclease.
- the cleaving enzyme is a phospho lipase
- the substrate moiety can comprise a diacylglycerolphosphate group recognized and cleaved by a particular phospholipase.
- sequences and structures recognized and cleaved by the various different types of cleaving enzymes are well known. Any of these sequences and structures can comprise the substrate moiety.
- the cleavage can be sequence specific, in some embodiments it can be non-specific.
- the cleavage can be achieved through the use of a non- sequence specific nuclease, such as, for example, an RNase.
- the substrate moieties described herein are not cleavable by phospholipases.
- Cleavage of the substrate moiety by the corresponding cleaving enzyme can release the fluorescent moiety from the micelle, reducing or eliminating its quenching and producing a measurable increase in fluorescence.
- the enzyme or agent acts upon the substrate moiety by the addition, deletion, or substitution of chemical moieties to the substrate moiety. These reactions can destabilize the substrate moiety in the micelle, thereby promoting its release from the micelle. The release of the substrate moiety increases the fluorescence of its fluorescent moiety.
- the enzyme or agent acts upon the substrate moiety to change the net charge of the substrate moiety, such as by phosphorylation of one or more unphosphorylated residues by a kinase enzyme or dephosphorylation of one or more phosphorylated residues by a phosphatase enzyme.
- substrate moieties modifiable by protein kinase and phosphatase enzymes are described in more detail below.
- the substrate moiety is first discussed below with reference to protein kinases as exemplary enzymes to be detected, quantified, and/or characterized.
- protein kinases are also useful for illustrating enzymes that cause an increase in the net charge of an substrate moiety by adding a phosphate group to a hydroxyl group to form a phosphorylated substrate moiety.
- phosphorylation of the substrate moiety causes the addition of two negative charges, for a net change in charge of " 2.
- Enzymes that carry out the opposite reaction, protein phosphatases are also discussed, which cause a net increase in charge of + 2 in the substrate moiety, under physiological conditions, i.e.
- the amplitude of the net charge on the substrate moiety is increased.
- the amplitude of the net negative charge on the substrate moiety is increased by " 2.
- the amplitude of the net positive charge on the substrate moiety is increased by + 2.
- a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into a micelle, a substrate moiety comprising a protein kinase recognition moiety comprising a consensus sequence including an unphosphorylated residue that is capable of being phosphorylated by a protein kinase, a fluorescent moiety and a charge-balance moiety is provided, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH.
- a micelle can comprise (i) a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into the micelle, a substrate moiety comprising a protein kinase recognition moiety comprising a consensus sequence including an unphosphorylated residue that is capable of being phosphorylated by a protein kinase; (ii) a fluorescent moiety; and, (iii) a charge-balance molecule comprising a hydrophobic moiety capable of integrating the charge-balance molecule into the micelle, and a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH.
- the fluorescent moiety can be part of the substrate molecule, the charge-balance molecule, or both.
- the protein kinase recognition moiety generally comprises an amino acid side chain containing a group that is capable of being phosphorylated by a protein kinase.
- the phosphorylatable group is a hydroxyl group.
- the hydroxyl group is provided as part of a side chain in a tyrosine, serine, or threonine residue, although any other natural or non-natural amino acid side chain or other entity containing a phosphorylatable hydroxyl group can be used.
- the phosphorylatable group can also be a nitrogen atom, such as the nitrogen atom in the epsilon amino group of lysine, an imidazole nitrogen atom of histidine, or a guanidinium nitrogen atom of arginine.
- the phosphorylatable group can also be a carboxyl group in an asparate or glutamate residue.
- Exemplary classes of protein kinases include cAMP-dependent protein kinases (also called the protein kinase A family, A-proteins, or PKA' s), cGMP -dependent protein kinases, protein kinase C enzymes (PKCs, including calcium dependent PKCs activated by diacylglycerol), Ca 2+ /calmodulin-dependent protein kinase I or II, protein tyrosine kinases (e.g., PDGF receptor, EGF receptor, and Src), mitogen activated protein (MAP) kinases (e.g., ERKl, KSSl, and MAP kinase type I), cyclin-dependent kinases (CDk's, e.g., Cdk2 and Cdc2), and receptor serine kinases (e.g., TGF-/3).
- cAMP-dependent protein kinases also called the protein kinase A family, A-protein
- Exemplary consensus sequences and/or enzyme substrates for various protein kinases are shown in Table 3, below. As will be appreciated by a person skilled in the art, these various consensus sequences and enzyme substrates can be used to design protein kinase recognition moieties having desired specificities for particular kinases and/or kinase families.
- Protein kinase recognition moieties having desired specificities for particular kinases and/or kinase families can also be designed, for example, using the methods and/or exemplary sequences described in Brinkworth et al., Proc. Natl. Acad. Sci. USA 100(1): 74- 79 (2003).
- the protein kinase recognition moieties comprise a sequence of L-amino acid residues.
- any of a variety of amino acids with different backbone or sidechain structures can also be used, such as: D-amino acid polypeptides, alkyl backbone moieties joined by thioethers or sulfonyl groups, hydroxy acid esters (equivalent to replacing amide linkages with ester linkages), replacing the alpha carbon with nitrogen to form an aza analog, alkyl backbone moieties joined by carbamate groups, polyethyleneimines (PEIs), and amino aldehydes, which result in polymers composed of secondary amines.
- PEIs polyethyleneimines
- the substrate moiety comprises one or more enzyme recognition moieties that can be acted upon by enzymes or agents. In some embodiments, the substrate moiety comprises one recognition moiety.
- the substrate moiety comprises two, three, four, or more recognition moieties.
- the recognition moieties can be the same or different.
- the recognition moieties can be connected in any way that permits them to perform their respective function.
- the recognition moieties can be directly connected to each other.
- the recognition moieties can be indirectly connected to each other via one or more linkage groups.
- the recognition moieties are indirectly linked to each other through the fluorescent moiety or the hydrophobic moiety.
- the recognition moiety includes all or a subset of the residues comprising a substrate or a consensus sequence for a specified enzyme.
- N the total number of residues comprising the consensus sequence is defined by N, wherein N is an integer from 1 to 10.
- N is an integer from 1 to 15.
- N is an integer from 1 to 20.
- the recognition moiety comprises a subset of residues comprising the consensus sequence for a specified enzyme.
- one or more residues are omitted from the consensus sequence.
- a subset is defined herein as comprising N- u amino acid residues, wherein, as defined above, N represents the total number of amino acid residues comprising the consensus sequence, and u represents the number of amino acid residues omitted from the consensus sequence.
- u is an integer from 1 to 9.
- u is an integer from 1 to 14.
- u is an integer from 1 to 19. For example, if the total number of amino acids in the consensus sequence is 4, subsets comprising 3, 2, or 1 amino acid residue(s) can be made.
- the total number of amino acids in the consesus sequence is 5, subsets comprising 4, 3, 2, or 1 amino acid residue(s) can be made. If the total number of amino acids in the consensus sequence is 6, subsets comprising 5, 3, 2, or 1 amino acid residue(s) can be made. If the total number of amino acids in the consensus sequence is 7, subsets comprising 6, 5, 4, 3, 2, or 1 amino acids residue(s) can be made. If the consensus sequence comprises 8 amino acids, subsets comprising 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) can be made. If the total number of amino acids in the consensus sequence is 9, subsets comprising 8, 7, 6, 5, 4, 3, 2, or 1 amino acids residue(s) can be made. If the consensus sequence comprises 10 amino acids, subsets comprising 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids residue(s) can be made. Typically, subsets comprising N-I or N-2 amino acid residues are made.
- two or more recognition moieties can share one or more residues comprising a consensus sequence.
- residues from one recognition moiety are included in another recognition moiety.
- the consensus sequence for protein kinase p38/3II is P-X-S-P.
- Two recognition moieties comprising one overlapping residue can be created, wherein one residue of the first substrate moiety P-X-S-P is shared with the second recognition moiety X-S-P, such that two recognition moieties, each comprising the consensus sequence for p38j3II is created, i.e., P-X-S-P-X-S-P.
- the number of residues to include in the consensus sequence depends on the specificity of the enzyme. For example, some protein kinases, such as p38/3II, require all of the residues comprising the consensus sequence to be present for phosphorylation activity to occur. Other protein kinases, such as PKC, can phosphorylate a consensus sequence, in which one or more residues are omitted from the consensus sequence.
- a substrate moiety comprising two or more recognition moieties, each recognition moiety comprising N residues for a given enzyme can be selected.
- a substrate moiety comprising two or more recognition moieties, one comprising a consensus sequence with N residues and the other comprising a consensus sequence with N-u residues can be selected.
- a substrate moiety comprising three, four, or more recognition moieties, each including consensus sequences comprising N residues for a given protein kinase can be selected.
- some of the recognition moieties can include consensus sequences with N residues, while other recognition moieties can include consensus sequences with N-u resides.
- substrate moieties comprising any combination of recognition moieties comprising N and N-u consensus sequences can be used, provided there is a detectable increase in fluorescence when the protein kinase is present.
- the recognition moieties can be for the same protein kinase, or they can be for different protein kinases. [0102] The distance between unphosphorylated residues depends, in part, on the location of the unphosphorylated residue(s) in a consensus sequence, and, in part, on the way in which the recognition moieties comprising the consensus sequences are connected.
- Unphosphorylated residues capable of being phosphorylated by a protein kinase can be adjacent, or they can be separated by one, two, three, or more residues that are not phosphorylated by a protein kinase.
- a substrate moiety comprising two unphosphorylated residues separated by three residues can be formed by connecting two recognition moieties, each comprising the consensus sequence -S-X-X-X- directly to each other to form a substrate moiety having the sequence -S-X-X-X— S-X-X-X-.
- a substrate moiety in which the unphosphorylated residues are separated by two residues can be formed by connecting two recognition moieties, one comprising a consensus sequence -P-X-S-P- and the other comprising an N-u, consensus sequence -X-S-P to form a substrate moiety having the sequence -P-X-S-P-X-S-P-.
- any combination of N and N- u consensus sequences, in which the unphosphorylated residues are adjacent, or are separated by one or more residues can be used in the kinase substrates provided that an increase in fluorescence is observed in the presence of the protein kinase(s).
- a consensus sequence comprising a phosphorylated residue can be designed for use with protein kinases that require a phosphorylated residue in order to phosphorylate one or more unphophosphorylated residue.
- GSK kinases are examples of kinases that require prior phosphorylation of a residue, before sequentially phosphorylating additional unphosphorylated residues (see Dajani, R. et al., 2001, Cell, 105: 721-732).
- one of the consensus sequences comprises a phosphorylated residue at one end, i.e., S(PO 4 2" ), T(PO 4 2" ), or Y(PO 4 2" ), and a unphosphorylated residue, i.e., S, T, or Y, at the opposite end.
- the phosphorylated residue can be separated from the unphosphorylated residue by one, two, three, four or more residues.
- the second consensus sequence can comprise N or N-u residues, and can be directly linked to the first consensus sequence or indirectly linked via a linker.
- a first consensus sequence comprising N residues, -S-X-X-X-S(PO 4 2" ), can be linked to a second consensus sequence comprising N-u residues, -S-X-X-X-, to provide a substrate moiety having the structure:
- the recognition moieties can be connected in any way that permits them to perform their respective function.
- the recognition moieties can be directly connected to each other.
- the recognition moieties can be indirectly connected to each other via one or more linkage groups.
- the recognition moieties are indirectly linked to each other through the fluorescent moiety, the charge balance moiety, or the hydrophobic moiety. Linkage groups suitable for indirectly connecting the various moieties described herein are discussed below.
- the recognition moieties can comprise a polypeptide segment containing the consensus sequence and additional residues (in addition to the phosphorylatable residue) that impart identifying features to the substrate to make it compatible with the substrate specificity of the protein kinase(s) to be detected, quantified, and/or characterized.
- the polypeptide segment has a polypeptide length equal to or less than 30 amino acid residues, 25 residues, 20 residues, 15 residues, 10 residues, or 5 residues.
- the polypeptide segment can have a polypeptide length in a range of 3 to 30 residues, or 3 to 25 residues, or 3 to 20 residues, or 3 to 15 residues, or 3 to 10 residues, or 3 to 5 residues, or 5 to 30 residues, or 5 to 25 residues, or 5 to 20 residues, or 5 to 15 residues, or 5 to 10 residues, or 10 to 30 residues, or 10 to 25 residues, or 10 to 20 residues, or 10 to 15 residues.
- the polypeptide segment contains at least 3, 4, 5, 6 or 7 amino acid residues.
- a phosphatase substrate moiety for detecting, quantifying, and/or characterizing one or more protein phosphates in a sample.
- a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into a micelle, a substrate moiety comprising a phosphatase recognition moiety comprising a phosphorylated residue that is capable of being dephosphorylated by a phosphatase, a fluorescent moiety and a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH, is provided.
- a micelle comprising (i) a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into the micelle, a substrate moiety comprising a phosphatase recognition moiety comprising at least one phosphorylated residue that is capable of being dephosphorylated by a phosphatase; (ii) a fluorescent moiety; and, (iii) a charge-balance molecule that comprises a hydrophobic moiety capable of integrating the charge-balance molecule into the micelle, a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH is provided.
- the fluorescent moiety can be part of the substrate molecule, the charge-balance molecule, or both.
- the phosphatase to be detected or characterized can be any phosphatase known in the art.
- the phosphate can be a phosphatase 2C, an alkaline phosphatase, or a tyrosine phosphatase.
- the phosphatase can be a phosphatase candidate, and the methods used to confirm and/or characterize the phosphatase activity of the candidate.
- Serine/threonine protein phosphatases represent a large class of enzymes that reverse the action of protein kinase A enzymes, for example.
- the serine/threonine protein phosphatases have been divided among four groups designated I, ILA, ILB, and IIC.
- Protein tyrosine kinases are also an important class of phosphatases, and histidine, lysine, arginine, and aspartate phosphatases are also known (e.g., see PJ. Kennelly, Chem Rev. 101 :2304-2305 (2001) and references cited therein).
- phosphatases are highly specific for only one or a few proteins, but in other cases, phosphatases are relatively non-specific and can act on a large range of protein targets. Accordingly, the phosphatase substrates of the present teachings can be designed to detect particular phosphatases by suitable selection of the phosphatase recognition moiety.
- peptide sequences that can be dephosphorylated by phosphatase activity are described in PJ. Kennelly, Chem. Rev. 101:2291-2312 (2001). Any of the exemplary consensus sequences and enzyme substrates shown in Table 3, can be used to design phosphatase substrate moieties having desired specificities for particular phosphatase and/or phosphatase families, provided that at least one residue is phosphorylated.
- the phosphatase recognition moiety can be designed to be reactive with a particular phosphatase or a group of phosphatases, or it can be designed to determine substrate specificity and other catalytic features, such as determining a value for kcat or Km.
- the phosphorylated residue in the phosphatase recognition moiety can be any group that is capable of being dephosphorylated by a phosphatase.
- the residue is a phosphotyrosine residue.
- the residue is a phosphoserine residue.
- the residue. is a phosphothreonine residue.
- the phosphatase recognition moiety can include additional amino acid residues (or analogs thereof) that facilitate binding specificity, affinity, and/or rate of dephosphorylation by the phosphatase.
- a substrate moiety comprising a sulfatase recognition for detecting or characterizing one or more sulfatases in a sample.
- a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into a micelle, a substrate moiety comprising a recognition moiety comprising a sulphate ester that is capable of being desulfated by a sulfatase, a fluorescent moiety, and a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH is provided.
- a micelle comprising (i) a substrate molecule comprising a hydrophobic moiety capable of integrating the substrate molecule into the micelle, a substrate moiety comprising a recognition moiety comprising a sulphate ester that is capable of being desulfated by a sulfatase; (ii) a fluorescent moiety; and, (iii) a charge-balance molecule that comprises a hydrophobic moiety capable of integrating the charge-balance molecule into the micelle, a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH is provided.
- the fluorescent moiety can be part of the substrate molecule, charge-balance molecule, or both.
- the sulfatase to be detected can be any sulfatase known in the art.
- the sulfatase is a 6-sulfate sulfatase, galactose-6-sulfate sulfatase, galNAc ⁇ S sulfatase, chondroitinsulfatase, and chondroitinase.
- the sulfatase can be a sulfatase candidate, and the method is used to confirm and/or characterize the sulfatase activity of the candidate.
- sulfatases A wide variety of sulfatases have been identified. In some cases, sulfatases are highly specific for only one or a few substrates, but in other cases, sulfatases are relatively non ⁇ specific and can act on a large range of substrates including, but not limited to, proteins, glycosaminoglycans, sulfolipids, and steroid sulfates.
- arylsulphatase A (EC: 3.1.6.8) (ASA), a lysosomal enzyme which hydrolyzes cerebroside sulphate; arylsulphatase B (EC: 3.1.6.12) (ASB), which hydrolyzes the sulphate ester group from N-acetylgalactosamine 4-sulphate residues of dermatan sulphate; arylsulphatase C (ASD) and E (ASE); steryl- sulphatase (EC: 3.1.6.2) (STS), a membrane bound microsomal enzyme which hydrolyzes 3- beta-hydroxy steroid sulphates; iduronate 2-sulphatase precursor (EC: 3.1.6.13) (IDS), a lysosomal enzyme that hydrolyzes the 2-sulphate groups from non-reducing-terminal iduronic acid residues in dermatan sulphate and heparan sulphate; N-acetyls
- compositions can be designed to detect particular sulfatases by selection of the sulfatase substrate moiety.
- Exemplary sulfatases and sulfatase substrates are shown in Table 4, below. These substrates can be used to design sulfatase recognition moieties having desired specificities for particular sulfatases and/or sulfatase families.
- the sulfatase recognition moiety can be designed to be reactive with a particular sulfatase or a group of sulfatases, or it can be designed to determine substrate specificity and other catalytic features, such as determining a value for kcat or Km.
- the sulphate ester in the sulfatase recognition moiety can be any group that is capable of being desulfated by a sulfatase.
- the sulfatase recognition moiety can include additional groups, for example amino acid residues (or analogs thereof) that facilitate binding specificity, affinity, and/or rate of desulfated by the sulfatase.
- a substrate moiety for detecting, quantifying and/or characterizing one or more protein peptidases in a sample comprising a hydrophobic moiety capable of integrating the substrate molecule into a micelle, a substrate moiety comprising a recognition moiety comprising a peptide bond capable of being hydrolyzed by a peptidase, a fluorescent moiety and a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 physiological pH is provided.
- a micelle comprising (i) a substrate molecule that comprises a hydrophobic moiety capable of integrating the substrate molecule into the micelle, a substrate moiety comprising a recognition moiety comprising a peptide bond capable of being hydrolyzed by a peptidase; (ii) a fluorescent moiety; and, (iii) a charge-balance molecule that comprises a hydrophobic moiety capable of integrating the charge-balance molecule into the micelle, a charge-balance moiety capable of balancing the overall charge of the micelle, such that the net charge of the micelle ranges from " 1 to + 1 at physiological pH is provided.
- the fluorescent moiety can be part of the substrate molecule, charge-balance molecule, or both.
- a peptidase is any member of a subclass of enzymes of the hydrolase class that catalyze the hydrolysis of peptide bonds. Generally, peptidases are divided into exopeptidases that act only near a terminus of a polypeptide chain and endopeptidases that act internally in polypeptide chains.
- the peptidase to be detected can be any peptidase known in the art. Also, the peptidase can be a peptidase candidate, and the methods used to confirm and/or characterize the peptidase activity of the candidate.
- peptidases A wide variety of peptidases have been identified. Generally, peptidases are classified according to their catalytic mechanisms: 1) serine peptidases (such as such as chymotrypsin and trypsin); 2) cysteine peptidases (such as papain); 3) aspartic peptidases (such as pepsin); and, 4) metallo peptidases (such as thermolysin).
- serine peptidases such as such as chymotrypsin and trypsin
- cysteine peptidases such as papain
- aspartic peptidases such as pepsin
- metallo peptidases such as thermolysin
- compositions can be designed to detect particular peptidases by suitable selection of the peptidase substrate moiety.
- -" are shown in Table 5, below. These various cleavage sites can be used to design peptidase substrate moieties having desired specificities for particular peptidases and/or peptidase families.
- Xaa - denotes any amino acid [0123]
- the peptidase recognition moiety can be designed to be reactive with a particular peptidase or a group of peptidases, or it can be designed to determine substrate specificity and other catalytic features, such as determining a value for kcat or Km.
- the peptidase recognition moiety can include additional amino acid residues (or analogs thereof) that facilitate binding specificity, affinity, and/or rate of hydrolysis by the peptidase.
- the substrate molecule and/or the charge-balance molecule can further comprise one or more charge-balance moiety(ies).
- the charge-balance moiety acts to balance the overall charge of the micelle.
- the substrate molecule comprises one or more charged chemical groups
- the presence of these groups can destabilize the substrate molecule in the micelle, thereby promoting the release of the substrate molecule from the micelle in the absence of the specified enzyme.
- Release of the charged substrate molecule from the micelle can be prevented by including a charge-balance molecule designed to counter the charge of the substrate molecule via the inclusion of chemical groups that have the opposite charge of the chemical groups comprising the substrate molecule, such that the overall charge of the micelle is approximately neutral.
- micelles can be formed in the presence of destabilizing chemical groups.
- FIG. 1 illustrates an exemplary embodiment of a single molecule embodiment of a substrate molecule comprising hydrophobic moiety R, a fluorescent moiety D, a substrate moiety S and a charge-balance moiety B.
- the fluorescence of the fluorescent moiety is quenched when the substrate molecule is incorporated into the micelle.
- the charge-balance moiety acts to balance the overall charge of the micelle such that micelle formation is promoted or encouraged.
- the hydrophobic moiety acts to integrate the substrate molecule(s) of the composition into a micelle when included in an aqueous solvent at or above its critical micelle concentration, thereby quenching the fluorescence fluorescent moiety.
- FIG. 2 illustrates an exemplary embodiment wherein the hydrophobic, fluorescent, substrate, and charge-balance moieties are included in two different distinct molecules.
- the substrate molecule comprises a hydrophobic moiety R, a fluorescent moiety D, and a substrate moiety S.
- the charge-balance molecule comprises a hydrophobic moiety R, a fluorescent moiety D, and a charge-balance moiety B. The fluorescence of the fluorescent moieties is quenched when the substrate molecule and charge-balance molecule are incorporated into the micelle.
- the charge-balance moiety act to balance the overall charge of the micelle such that micelle formation is promoted or encouraged.
- the hydrophobic moieties act to integrate the substrate molecule and the charge-balance molecule of the composition into a micelle when included in an aqueous solvent at or above the critical micelle concentration, thereby quenching the fluorescence of the fluorescent moieties.
- FIG. 3 illustrates an exemplary embodiment wherein the hydrophobic, fluorescent, substrate, charge-balance moieties, and a quenching moiety are included in three different distinct molecules.
- the quenching molecule comprises a quenching moiety and a hydrophobic moiety.
- the hydrophobic moiety integrates the quenching molecule into the micelle.
- the quenching moiety is selected such that it is capable of quenching the fluorescence of a fluorescent moiety of the molecule(s) of the compositions comprising the micelle. If the micelle comprises a plurality of molecules having different fluorescent moieties, a quenching moiety capable of quenching the fluorescence of all or a subset of the fluorescent moieties can be selected. Any of the hydrophobic and quenching moieties previously described can be used to construct a quenching molecule. In other embodiments, the quenching moiety can be part of the substrate molecule or the charge-balance molecule.
- the substrate molecule comprises a hydrophobic moiety R, a fluorescent moiety D, and a substrate moiety S.
- the charge-balance molecule comprises a hydrophobic moiety R, a fluorescent moiety D, and a charge-balance moiety B.
- the quenching molecule comprises a hydrophobic moiety R and a quenching moiety Q. The fluorescence of the fluorescent moieties is quenched when the substrate molecule, charge-balance molecule, and quenching molecule are incorporated into the micelle.
- the charge-balance moiety act to balance the overall charge of the micelle such that micelle formation is promoted or encouraged.
- the hydrophobic moieties act to integrate the substrate molecule, the charge- balance molecule, and the quenching molecule of the composition into a micelle when included in an aqueous solvent at or above the critical micelle concentration, thereby quenching the fluorescence of the fluorescent moiety.
- the molar ratio of quenching moiety to fluorescent moiety can be any ratio capable of quenching the fluorescent moiety in the micelle. In some embodiments, the molar ratio between the quenching moiety and fluorescent moiety is 1 to 1. In other embodiments, the molar ratio between the quenching moiety and fluorescent moiety is 1 to 2. In other embodiments the molar ratio between the quenching moiety and fluorescent moiety is 1 to 5, or 1 to 10. In some embodiments, the molar ratio between the fluorescent moiety and quenching moiety is 1 to 2. In other embodiments the molar ratio between the fluorescent moiety and quenching moiety is 1 to 5, or 1 to 10.
- the charge-balance moiety can be designed to balance the overall charge of the micelle such that net charge of the micelle is about neutral.
- the overall charge of the micelle depends in part on a number of factors including its chemical composition and pH of the solution comprising the micelle.
- the substrate molecule comprises a florescent moiety and a substrate moiety, both of which comprise one ore more charged chemical groups that can destabilize or prevent micelle formation.
- a charge-balance molecule that is capable of countering the charge of the substrate molecule, micelles with a net charge between " 1 to + 1 can be formed at a pH on the range of 6 to 8.
- the charge of the charge-balance molecule depends in part, on the presence of the other charged groups comprising the micelle.
- the charge-balance molecule can be designed to have a net negative or net positive charge by including an appropriate number of negatively and positively charged groups in the charge-balance moiety.
- a net positive charge i.e., net charge + 2
- the charge-balance moiety can be designed to contain positively charged groups, or a greater number of positively charged groups than negatively charged groups.
- a net negative charge i.e., net charge " 2
- the charge-balance moiety can be designed to contain negatively charged groups, or a greater number of negatively charged groups than positively charged groups.
- the overall charge of the charge-balance molecule also depends in part upon other factors such as the molar ratio of the substrate molecule:charge-balance molecule, the pH of the assay medium, and concentration of salt in the assay medium.
- the ratio of charge-balance molecule to substrate molecule can be any ratio capable of balancing the overall charge of the micelle.
- the molar ratio between the charge-balance molecule and substrate molecule is 0.5 to 1.
- the molar ratio between the charge-balance molecule and substrate molecule is 1 to 1.
- the molar ratio between the charge-balance molecule and substrate molecule is 1 to 2, or 1 to 5, or 1 to 10.
- the molar ratio between the substrate molecule and charge-balance molecule is 1 to 1.
- the molar ratio between the substrate molecule and charge-balance molecule is 1 to 2.
- the molar ratio between the substrate molecule and charge- balance molecule is 1 to 2, or 1 to 5, or 1 to 10.
- the + 2 charge can be balanced by adding an equal molar ratio of a charge-balance molecule with a net charge of " 2.
- the charge can be balanced by adding a charge-balance molecule with a net charge of " 1 at a 1 :2 molar ratio of substrate molecule to charge-balance molecule.
- Another factor effecting the charge of the charge-balance moiety is the pH of the assay medium and the pKas' of the groups comprising the charge-balance moiety.
- the charge-balance moiety is designed to carry a positive charge at pH 7.6, then amino acids with side chains having pKas' above 7.6 can be chosen i.e. lysine (pKa 10.5) and arginine (pKa 12.5) carry a positive charge at pH 7.6.
- amino acids with side chains having pKas' below 7.6 can be chosen i.e.
- the charge-balance moiety comprises any group capable of carrying a charge. Suitable examples include amino acids, amino acid analogs, and derivatives, and quartenary compounds such as ammonium and amine compounds.
- the charge-balance moiety can comprise positively charged amino acids such as arginine and lysine. Lysine and arginine contain side chains that carry a single positive charge at physiological pH.
- the imidazole side chain of histidine has a pKa of about 6, so it carries a full positive charge at a pH of about 6 or less.
- the charge-balance moiety can comprise negatively charged amino acids such as aspartic acid and glutamic acid.
- Aspartic acid and glutamic acid contain carboxyl side chains having a single negative charge. Cysteine has a pKa of about 8, so it carries a full negative charge at a pH above 8.
- the charge-balance moiety can comprise a phosphorylated amino acid. For example, a phosphoserine residue carries two negative charges on a phosphate group.
- the charge-balance moiety can comprise uncharged amino acids such as alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine (physiological pH 6 to 8).
- uncharged amino acids such as alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine (physiological pH 6 to 8).
- the charge-balance moiety can comprise uncharged amino acids analogs. Suitable examples include 2-amino-4-fluorobenzoic acid, 2-amino-3- methoxybenzoic acid, 3,4-diaminobenzoic acid, 4-aminomethyl-L-phenylalanine, 4-bromo- L-phenylalanine, 4-cyano-L-proline, 3,4,-dihydroxy-L-phenylalanine, ethyl-L-tyrosine, 7- azaatryptophan, 4-aminohippuric acid, 2 amino-3-guanidinopropionic acid, L-citrulline, and derivatives.
- the charge-balance moiety can comprise positively charged amino acids analogs such as N- ⁇ , ⁇ -dimethyl-L-arginine, a-methyl-DL-orni thine, N- ⁇ -nitro- L- arginine, and derivatives.
- the charge-balance moiety can comprise negatively charged amino acids analogs such as 2-aminoadipic acid, N-a-(4-aminobenzoyl)-L-glutamic acid, iminodiacetic acid, a-methyl-L-aspartic acid, a-methyl-DL-glutamic acid, y-methylene-DL- glutamic acid, and derivatives.
- negatively charged amino acids analogs such as 2-aminoadipic acid, N-a-(4-aminobenzoyl)-L-glutamic acid, iminodiacetic acid, a-methyl-L-aspartic acid, a-methyl-DL-glutamic acid, y-methylene-DL- glutamic acid, and derivatives.
- the various moieties described herein can be connected in any way that permits them to perform their respective functions.
- the various moieties can be connected directly to one another, i.e., covalently linked to each other.
- one, some or all of the moieties can be connected indirectly to one another, i.e., via one or more optional linkers.
- a linker having properties suitable for a particular application is within the capabilities of those having skill in the art.
- a rigid linker may comprise a rigid polypeptide such as polyproline, a rigid polyunsaturated alkyldiyl or an aryldiyl, biaryldiyl, arylarydiyl, arylalkyldiyl, heteroaryldiyl, biheteroaryldiyl, heteroarylalkyldiyl, heteroaryl-heteroaryldiyl, etc.
- a flexible linker may comprise a flexible polypeptide such as polyglycine or a flexible saturated alkanyldiyl or heteroalkanyldiyl.
- Hydrophilic linkers may comprise, for example, polyalcohols or polyethers such as polyalkyleneglycols, and O-spacers, as described below.
- Hydrophobic linkers may comprise, for example, alkyldiyls or aryldiyls.
- any molecule having three or more "reactive" groups suitable for attaching other molecule and moieties thereto, or that can be appropriately activated to attach other molecules and moieties thereto could be used to provide a trivalent or higher order multivalent linker.
- the "backbone" of the multivalent linker to which the reactive linking groups are attached could be linear, branched or cyclic saturated or unsaturated alkyl, a mono or polycyclic aryl or an arylalkyl.
- the previous examples are hydrocarbons, the multivalent linker backbone need not be limited to carbon and hydrogen atoms.
- a multivalent linker backbone can include single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon- oxygen bonds, carbon-sulfur bonds and combinations thereof, and therefore can include functionalities such as carbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas, urethanes, hydrazines, etc.
- linkers comprised of stable bonds that are suitable for use in the substrates described herein are known in the art, and include by way of example and not limitation, alkyldiyls, substituted alkyldiyls, alkylenos (e.g., alkanos), substituted alkylenos, heteroalkyldiyls, substituted heteroalkyldiyls, heteroalkylenos, substituted heteroalkylenos, acyclic heteroatomic bridges, aryldiyls, substituted aryldiyls, arylaryldiyls, substituted arylaryldiyls, arylalkyldiyls, substituted arylalkyldiyls, heteroaryldiyls, substituted heteroaryldiyls, heteroaryl-heteroaryl diyls, substituted heteroaryl-heteroaryl diyls, heteroarylalkyldiy
- the linker can include single, double, triple or aromatic carbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen bonds, carbon- oxygen bonds, carbon-sulfur bonds and combinations of such bonds, and may therefore include functionalities such as carbonyls, ethers, thioethers, carboxamides, sulfonamides, ureas, urethanes, hydrazines, etc.
- the linker comprises from 1-20 non- hydrogen atoms selected from the group consisting of C, N, O, and S and is composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamides, hydrazide, aromatic and heteroaromatic groups.
- linkages are formed from pairs of complementary reactive groups capable of forming covalent linkages with one another.
- “Complementary” nucleophilic and electrophilic groups or precursors thereof that can be suitable activated) useful for effecting linkages stable to biological and other assay conditions are well known. Examples of suitable complementary nucleophilic and electrophilic groups, as well as the resultant linkages formed therefrom, are provided in Table 7.
- *Activated esters as understood in the art, generally have the formula -C(O)Z, where Z is, a good leaving group (e.g., oxysuccinimidyl, oxysulfosuccinimidyl, 1-oxybenzotriazolyl, etc.). **Acyl azides can rearrange to isocyanates.
- FIGS. 4A-I, 5, 6, 7A-D, and 8A-H Exemplary embodiments illustrating various combinations for linking the different moieties are provided in FIGS. 4A-I, 5, 6, 7A-D, and 8A-H.
- FIGS. 4A-E illustrate exemplary embodiments of substrate molecules comprising a substrate molecule comprising a hydrophobic moiety, illustrated as R 1 -C(O)-, two or more recognition moieties for a protein kinase, illustrated as (PKRM), and a fluorescent dye.
- a substrate molecule comprising a hydrophobic moiety, illustrated as R 1 -C(O)-, two or more recognition moieties for a protein kinase, illustrated as (PKRM), and a fluorescent dye.
- PKRM protein kinase
- the C-terminus of the first kinase recognition moiety is linked to the N-terminus of the second protein kinase recognition moiety via a peptide bond.
- the illustrated hydrophobic moiety, R 1 can comprise any of the hydrophobic groups described above.
- R 1 can comprise saturated or unsaturated alkyl chains, which may be the same or different.
- the hydrophobic moiety Ri- C(O)- is linked to the fluorescent moiety via an optional linker L 1 .
- the fluorescent moiety represented by Dye, can be linked to the protein kinase recognition moieties, directly or indirectly via an optional linker.
- the presence or absence of optional linkers, such as L 1 is denoted by the value for q, which may 0 or 1.
- the fluorescent moiety is linked directly to the protein kinase recognition moieties.
- Optional linkers, such as L 1 can be any of the linkage groups described herein, but are typically provided by one or more (bis)ethylene glycol group(s), also referred to herein as an "O- spacer".
- the hydrophobic moiety Ri- C(O)- is linked to the fluorescent moiety via an O-spacer, i.e. optional linker 10.
- O-spacer i.e. optional linker 10.
- the value of m can range broadly, but is typically an integer from 0 to 6.
- each "O-spacer" corresponds to the bracketed illustrated structure.
- the substrate is referred to herein as comprising three O-spacers (which can be abbreviated as "O-O-O").
- the O- spacer comprises n oxyethylene units.
- n is an integer from 1 to 10. In other embodiments, n is 1, 2, 3, 4, 5 or 6.
- an O-spacer need not be composed of oxyethylene units. Virtually any combination of the same or different oxyethylene units that permits the substrate to function as described herein may be used. In a specific example, an O-spacer may comprise from 1 to about 5 of the same or different lower oxyethylene units ⁇ e.g., -(CH 2 ) X CH 2 )-, where x is an integer ranging from 0 to 6).
- the protein kinase recognition moieties are not connected directly to each other.
- the kinase recognition moieties can be connected to each other via optional linker L 2 .
- L 2 can be an of the linkage groups described herein, but is typically provided by one or more "O-spacers". Additional exemplary embodiments of kinase substrates are illustrated in FIGS. 4D and 4E.
- FIG. 4D illustrates an exemplary kinase substrate that is similar to the kinase substrate depicted in FIG. 4A, with the exception that the hydrophobic moiety is linked to the fluorescent moiety and the terminal NH 2 group via a multivalent (trivalent) linker, which in the specific embodiment illustrated in FIG. 4D is provided by the amino acid lysine. Similarly, the fluorescent moiety is linked to the hydrophobic moiety and optional linker 10 wo a trivalent linker provided by the amino acid lysine.
- FIG. 4E illustrates an exemplary kinase substrate that is similar to the kinase substrate depicted in FIG. 4D, with the exception that exemplary kinase substrate depicted in FIG.
- 4E comprises three protein kinase recognition moieties.
- the first two protein kinase recognition moieties are directly connected to each other, and indirectly connected to the third protein kinase recognition moiety via an optional linkage group.
- multivalent linkers can also be used to connect the recognition moieties to each other, to the hydrophobic moiety, and/or to the fluorescent moiety
- the kinase substrate molecule includes two hydrophobic moieties.
- the fluorescent moiety (Dye-C(O)- is linked to the first hydrophobic moiety and the N-terminal end of the first protein kinase recognition moiety via a multivalent (trivalent) linker, which in the specific embodiment illustrated in FIG. 4F is provided by the amino acid lysine.
- the second hydrophobic moiety, represented by R 2 -C(O)- is linked the C-terminal end of the second protein kinase recognition moiety. As illustrated, the linkage, is spaced away from the C-terminus of the second protein recognition sequence via optional linker L 2 .
- optional linkers L 1 and L 2 are used to connect the first and second hydrophobic moieties to the fluorescent moiety and to the protein kinase recognition moieties.
- Optional linkers L 1 and L 2 can comprise any of the various atoms and groups discussed above in connection with optional linker 10.
- Optional linkers L 1 and L 2 may both be present, they may both be absent, or, alternatively, one of linkers L 1 and L 2 may be present and the other absent.
- FIG. 4G illustrates an exemplary kinase substrate molecule in which the two recognition moieties are indirectly connected to each other via optional linker L 2 .
- 4G illustrates an exemplary kinase substrate molcule in which the first hydrophobic moiety is connected either directly, or indirectly via optional linker L 1 to the N-terminus of the first protein kinase recognition moiety.
- the second hydrophobic moiety is linked to the fluorescent moiety and the terminal NH 2 group via a trivalent linker, which in the specific embodiment illustrated in FIG. 4G is provided by the amino acid lysine.
- the fluorescent moiety is linked to the hydrophobic moiety and the C-terminus of the second protein kinase recognition moiety via a trivalent linker provided by the amino acid lysine.
- FIG. 41 illustrates an exemplary embodiment of a kinase substrate molecule that comprises two fluorescent moieties. Although the two fluorescent moieties are illustrated as being the same, they could be different.
- FIG. 5 An exemplary kinase substrate, Ci 6 -OOOK(Dye2)LSPSLSRHSS(PO4 2" )HQRRR- NH 2 , comprising two protein kinase recognition sequences, i.e., SRHSS(PO4 2" ) and SPSLS for GSK is illustrated in FIG. 5.
- C ⁇ -OOK(dye2)PJUPLSPLSPOOKC u -NH 2 comprising two protein kinase recognition sequences, i.e., -PLSP- and -PLSP- for p38/3II, is illustrated in FIG. 6.
- kinase substrate molcules illustrated in FIGS. 4A-4I, 5 and 6 are exemplified with different combinations of hydrophobic moieties, fluorescent moieties, protein kinase recognition sequences, phosphorylatable moieties, and optional linkers, any one or more of these features of the illustrated kinase substrates could be varied.
- the kinase substrate molecules are exemplified with one or more hydrophobic moieties, one or more fluorescent moieties, and a substrate moiety comprising two or more recognition moieties
- charge-balance moieties described below
- the substrate moiety can comprise one or more recognition moieties.
- FIGS. 7A-D illustrate exemplary embodiments wherein the hydrophobic, fluorescent, substrate, and charge-balance moieties are included in a single molecule.
- hydrophobic moiety R is connected to the remainder of the substrate molecule via a peptide linkage.
- the hydrophobic moiety R is linked to the remainder of the substrate molecule via an optional linker.
- R can comprise any of the hydrophobic moieties described above.
- the fluorescent moiety Dye is connected to the remainder of the substrate molecule via a ((CH 2 ) P -NH-CO-) linkage, wherein p can be any integer form 1 to 6.
- Substrate moiety X can comprise one or more of the recognition moieties described above.
- FIG. 7A illustrates an exemplary embodiment wherein the charge of the substrate moiety X is balanced by an opposite charge on the charge-balance moiety Y 1 .
- the charge of the fluorescent moiety Dye is balanced by an opposite charge on a second charge-balance moiety Y 2 .
- FIGS. 8A-H illustrate exemplary embodiments of compositions comprising two distinct molecules, a substrate molecule (i.e. FIGS. 8 A, C, E, G) and a charge-balance molecule (i.e. FIGS. 8B, D, F, H).
- a substrate molecule i.e. FIGS. 8 A, C, E, G
- a charge-balance molecule i.e. FIGS. 8B, D, F, H
- hydrophobic moiety R and substrate moiety X can comprise any of the hydrophobic moieties or recognition moieties described above.
- the substrate molecule and charge-balance molecule comprise the fluorescent moiety Dye.
- FIGS. 8A-B illustrate an exemplary embodiment of a composition comprising a substrate molecule and a charge-balance molecule, wherein fluorescent moiety Dye is connected to the substrate moiety X.
- the charge of the substrate moiety X in the substrate molecule illustrated in FIG. 8A can be balanced by an opposite charge on charge-balance moiety Yi in the charge-balance molecule illustrated in FIG. 8B.
- the charge of the fluorescent moiety Dye in the substrate molecule illustrated in FIG. 8A can be balanced by an opposite charge on charge-balance moiety Y 2 comprising the charge-balance molecule illustrated in FIG. 8B.
- FIGS. 8C-D illustrate an exemplary embodiment of a composition comprising a substrate molecule (FIG. 8C) and a charge-balance molecule (FIG. 8D), comprising a fluorescent moiety Dye and charge-balance moiety Yi.
- the charge of substrate moiety X in FIG. 8C is balanced by an opposite charge on charge-balance moiety Yi in FIG. 8D.
- the charge of fluorescent moiety Dye in FIG. 8D is balanced by an opposite charge on charge- balance moiety Y 2 in FIG. 8C.
- FIGS. 8E-F illustrate an exemplary embodiment of a composition comprising a substrate molecule (FIG. 8E) and a charge-balance molecule (FIG. 8F).
- the substrate molecule illustrated in FIG. 8E comprises a fluorescent moiety Dye, substrate moiety X and hydrophobic moiety R.
- the charge of substrate moiety X in FIG. 8E is balanced by an opposite charge on charge-balance moiety Yi in FIG. 8F.
- the charge of fluorescent moiety Dye in FIG. 8E is balanced by an opposite charge on charge-balance moiety Y ⁇ in FIG. 8F.
- FIGS. 8G-H illustrate an exemplary embodiment of a composition comprising a substrate molecule (FIG. 8G) and a charge-balance molecule (FIG. 8H).
- the substrate molecule illustrated in FIG. 8G comprises a charge balance moiety Y 2 , a substrate moiety X, and hydrophobic moiety R.
- the charge of substrate moiety X in the in FIG. 8G is balanced by an opposite charge on charge-balance moiety Yi in FIG. 8H.
- the charge of fluorescent moiety Dye in FIG. 8H is balanced by an opposite charge on charge-balance moiety Y 2 in FIG. 8G.
- R is a hydrophobic moiety; each s is, independently of the other, 0 or 1; q represents a linker, each q is, independently of the other, 0 or 1; m is an integer from 0 to 10; n is an integer from 0 to 10; r represents a fluorescent moiety, each r is, independently of the other, 0 or 1; each p is, independently of the other, an integer from 1 to 6; X comprises a substrate moiety; and Yi-Y 3 comprise charge-balance moieties.
- FIG. 9 illustrates exemplary embodiments of a substrate molecule (FIG. 9A) and a charge-balance molecule (FIG. 9B).
- FIG. 9A illustrates an exemplary substrate molecule that can be used to detect a protein kinase that recognizes a peptide consensus sequence for the tyrosine kinase Lyn, i.e.
- hydrophobic moiety is a Ci 6 carbon chain and the fluorescent moiety, 5-carboxy-2',7'-dipyridyl-sulfonefluorescein is linked to the hydrophobic moiety and an optional linker via the amino acid lysine.
- the recognition moiety comprises the peptide sequence Glu-Glu-Ile-Tyr-Gly-Glu-Phe.
- FIG. 9B illustrates an exemplary charge-balance molecule (i.e. C 16 ArgArgOOOArgArgIleTyrGlyArgPheNH 2 , wherein OOO represents the optional O- spacers) that can be used balance the charge of the substrate molecule illustrated in FIG. 9A.
- the substrate molecule illustrated in FIG. 9A comprises a fluorescent moiety containing a sulfonate anion with a charge of " 2.
- the substrate molecule illustrated in FIG. 9A further comprises a recognition moiety comprising three glutamate residues, each with a ⁇ 1 charge.
- the total negative charge of the substrate molecule illustrated in FIG. 9A is ⁇ 5 at physiological pH.
- FIG. 9B comprises guanidinium groups in the five arginine residues, each having a + 1 charge.
- the total positive charge of the charge-balance molecule illustrated in FIG. 9B is + 5 at pH 7.6.
- the net charge of the compound comprising the substrate molecule illustrated in FIG. 9A and the charge-balance molecule illustrated in FIG. 9B is approximately zero at pH 7.6.
- the net charge of the micelle comprising the substrate molecule and charge-balance molecule is changed from approximately zero to " 2, thereby promoting the dissociation of the fluorescent moiety from the micelle, thereby reducing or eliminating the quenching effect and producing a detectable increase in fluorescence.
- the substrate moiety comprises the amino acid sequence E-E-I-Y-G-E-F- (SEQ ID NO:32) and has a net charge of " 3 at pH 7.6, then the charge-balance moiety can comprise an amino acid sequence -R-R-E-I-Y-G-R-F- (SEQ ID NO:33) and has a net charge of + 3 at pH 7.6.
- any protein kinase sequence such as the various consensus sequences provided in Table 1, supra, may be used. Skilled artisans will be readily able to select a protein kinase consensus sequence suitable for a particular application.
- the various substrate and/or charge-balance molecules can comprise additional moieties.
- a substrate molecule can comprise a charge-balance moiety and vice-versa.
- the compositions can comprise a quenching moiety.
- the substrate molecules and charge-balance molecules can be readily prepared by synthetic methods known in the art.
- Polypeptides can be prepared by automated synthesizers on a solid support (Perkin J. Am. Chem. Soc. 85:2149-2154 (1963)) by any of the known methods, e.g. Fmoc or BOC (e.g., Atherton, J. Chem. Soc. 538-546 (1981); Fmoc Solid Phase Peptide Synthesis. A Practical Approach, Chan, Weng C. and White, Peter D., eds., Oxford University Press, New York, 2000).
- polypeptides can be formed by a condensation reaction between the ⁇ -carbon carboxyl group of one amino acid and the amino group of another amino acid. Activated amino acids are coupled onto a growing chain of amino acids, with appropriate coupling reagents.
- Polypeptides can be synthesized with amino acid monomer units where the ⁇ -amino group was protected with Fmoc (fiuorenylmethoxycarbonyl). Alternatively, the BOC method of peptide synthesis can be practiced to prepare the peptide conjugates of the present teachings.
- Amino acids with reactive side-chains can be further protected with appropriate protecting groups.
- Amino groups on lysine side-chains to be labelled can be protected with an Mtt protecting group, selectively removable with about 5% trifluoroacetic acid in dichloromethane.
- Mtt protecting group selectively removable with about 5% trifluoroacetic acid in dichloromethane.
- a large number of different protecting group strategies can be employed to efficiently prepare polypeptides.
- Exemplary solid supports include polyethyleneoxy/polystyrene graft copolymer supports (TentaGel, Rapp Polymere GmbH, Tubingen, Germany) and a low-cross link, high- swelling Merrifield-type polystyrene supports with an acid-cleavable linker (Applied Biosystems), although others can be used as well.
- Polypeptides are typically synthesized on commercially available synthesizers at scales ranging from 3 to 50 ⁇ moles.
- the Fmoc group is removed from the terminus of the peptide chain with a solution of piperidine in dimethylformamide (DMF), typically 30% piperidine, requiring several minutes for deprotection to be completed.
- DMF dimethylformamide
- the amino acid monomer, coupling agent, and activator are delivered into the synthesis chamber or column, with agitation by vortexing or shaking.
- the coupling agent is HBTU
- the activator is 1-hydroxybenzotriazole (HOBt).
- the coupling solution also can contain diisopropylethylamine or another organic base, to adjust the pH to an optimal level for rapid and efficient coupling.
- Peptides can alternatively be prepared on chlorotrityl polystyrene resin by typical solid-phase peptide synthesis methods with a Model 433 A Peptide Synthesizer (Applied Biosystems, Foster City, CA) and Fmoc/HBTU chemistry (Fields, (1990) Int. J. Peptide Protein Res. 35:161-214).
- the crude protected peptide on resin can be cleaved with 1% trifluoroacetic acid (TFA) in methylene chloride for about 10 minutes.
- TFA trifluoroacetic acid
- the filtrate is immediately raised to pH 7.6 with an organic amine base, e.g. 4-dimethylaminopyridine. After evaporating the volatile reagents, a crude protected peptide is obtained that can be labelled with additional groups.
- the peptide on the solid support is deprotected and cleaved from the support. Deprotection and cleavage can be performed in any order, depending on the protecting groups, the linkage between the peptide and the support, and the labelling strategy. After cleavage and deprotection, peptides can be desalted by gel filtration, precipitation, or other means, and analyzed. Typical analytical methods useful for the peptides and peptide conjugates of the present teaching include mass spectroscopy, absorption spectroscopy, HPLC, and Edman degradation sequencing. The peptides and peptide conjugates of the present teachings can be purified by reverse-phase HPLC, gel filtration, electrophoresis, or dialysis.
- Fluorescent dyes can be incorporated into the molecules described herein using methods known in the art.
- a fluorescent dye labeling reagent can bear an electrophilic linking moiety which reacts with a nucleophilic group on the polypeptide, e.g. amino terminus, or side-chain nucleophile of an amino acid.
- the dye can have a nucleophilic moiety, e.g. amino- or thiol- linking moiety, which reacts with an electrophilic group on the peptide, e.g. NHS of the carboxyl terminus or carboxyl side-chain of an amino acid.
- Fluorescent dyes that can be used to prepare the molecules can be prepared synthetically using conventional methods or purchased commercially (e.g. Sigma- Aldrich and/or Molecular Probes).
- Non-limiting examples of methods that can be used to synthesize suitably reactive fluorescein and/or rhodamine dyes can be found in the various patents and publications discussed above in connection with the fluorescent moiety.
- Non-limiting examples of suitably reactive fluorescent dyes that are commercially available from Molecular Probes (Eugene, OR) are provided in Table 8, below:
- compositions find a wide variety of uses in detecting, quantifying and/or characterizing enzymes in biological, medical and industrial applications.
- the methods generally comprise detecting, quantifying and/or characterizing enzymes in a sample with one or more molecules that collectivity include three to four different types of moieties: a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety.
- the sample to be tested can be any suitable sample selected by the user.
- the sample can be naturally occurring or man-made.
- the sample can be a blood sample, tissue sample, cell sample, buccal sample, skin sample, urine sample, water sample, or soil sample.
- the sample can be from a living organism, such as a eukaryote, prokaryote, mammal, human, yeast, or bacterium.
- the sample can be processed prior to contact with a substrate of the present teachings by any method known in the art.
- the sample can be subjected to a lysing step, precipitation step, column chromatography step, heat step, etc.
- the sample is a purified or synthetically prepared enzyme that is used to screen for or characterize an enzyme substrate, inhibitor, activator, or modulator.
- an inactivating agent e.g., an active site directed an irreversible inhibitor
- the reaction mixture typically includes a buffer, such as a buffer described in the "Biological Buffers" section of the 2000-2001 Sigma Catalog.
- a buffer such as a buffer described in the "Biological Buffers" section of the 2000-2001 Sigma Catalog.
- Exemplary buffers include MES, MOPS, HEPES, Tris (Trizma), bicine, TAPS, CAPS, and the like.
- the buffer is present in an amount sufficient to generate and maintain a desired pH.
- the pH of the reaction mixture is selected according to the pH dependency of the activity of the enzyme to be detected, and the charge of the various moieties described herein.
- the pH can be from 2 to 12, from 5 to 9, or from 6 to 8.
- the reaction mixture can also contains salts, reducing agents such as dithiothreitol (DTT), and any necessary cofactors and/or cosubstrates for the enzyme (e.g., ATP for a protein kinase, Ca 2+ ion for a calcium dependent kinase, and c AMP for a protein kinase A).
- DTT dithiothreitol
- the reaction mixture does not contain detergent or is substantially free from detergents.
- the sample to be tested can be diluted to any concentration that permits a detectable increase in fluorescence.
- the sample can be diluted 1, 2, 5, 10, 20, 30, 40, or 50-fold. In some embodiments, a greater 50-fold dilution of the sample can be desirable. In some embodiments the sample can be diluted in the assay reaction mixture.
- the ionic strength can be kept disfavored and destabilized.
- high salt concentration e.g., 1 M NaCl
- Guidance regarding the effects of ionic species, such as metal ions, can be found in Surfactants and Interfacial Phenomena, 2nd Ed., MJ. Rosen, John Wiley & Sons, New York (1989), particularly chapter 3. For example, Mg 2+ ion at a concentration of 5 mM is useful in the Examples provided below, but higher concentrations can give poorer results.
- a substrate molecule (or substrate molecule and charge-balance molecule) is mixed with a sample containing an enzyme that is to be detected or that is being used to screen for, detect, quantify, and/or characterize a compound for substrate, inhibitor, activator, or modulator activity.
- Reaction of the enzyme with the substrate molecule causes an increase (to a more charged species) in the absolute amplitude of the net charge of the micelle, such that the fluorescence of the reacted micelle is greater than the fluorescence of the unreacted micelle.
- the substrate molecule (or substrate molecule and charge-balance molecule) has a net charge of zero (neutral net charge), and reaction of the substrate molecule with the enzyme makes the substrate molecule either (1) net negatively charged by (IA) adding or generating a new negatively charged group on the recognition moiety, or (IB) removing or blocking a positively charged group on the recognition moiety; or (2) net positively charged, by (2A) adding or generating a new positively charged group on the recognition moiety, or (2B) removing or blocking a negatively charged group on the recognition moiety.
- reaction (IA) can be accomplished by adding a phosphate group to a hydroxyl group on the recognition moiety (changing a neutrally charged group to a group having a charge of -2, (e.g., using a protein kinase), by cleaving a carboxylic ester or amide to produce a carboxyl group (changing a neutrally charged group to a group having a charge of - 1, e.g., using an esterase or amidase).
- Reaction (IB) can be accomplished by cleaving a positively charge amino acids, or can be accomplished by reacting an amino or hydrazine group in the enzyme recognition moiety with an acetylating enzyme to produce a neutral acetyl ester group, with an N-oxidase enzyme to produce a neutral N-oxide, with an ammonia lyase to remove ammonia, or with an oxidase that causes oxidative deamination, for example.
- Reaction (2A) can be accomplished, for example, by treating an amide group in the enzyme recognition moiety with an amidase to generate a positively charged amino group in the substrate moiety.
- Reaction (2B) can be accomplished by cleaving a negativity charge amino acids, or can be accomplished using a decarboxylase enzyme to remove a carboxylic acid or by reacting a carboxyl group with a methyl transferase to form a carboxylic ester, for example.
- a decarboxylase enzyme capable of performing such transformations are known in the literature (e.g., see C. Walsh, Enzymatic Reaction Mechanisms. WH Freeman and Co., New York, (1979), the Worthington Product Catalog (Worthington Enzymes), Sigma Life Sciences Catalog, and the product catalogs of other commercial enzyme suppliers).
- FIGS. 5 and 6 illustrate exemplary kinase substrates.
- FIG. 5 illustrates an exemplary kinase substrate that can be used to detect a protein kinase that recognizes a peptide consensus sequence for protein kinase GSK, i.e., Ci 6 -OOO-Lys-(dye2)- LysSerProSerLysArgHisSerSer(PO 4 2" )HisGlnArgArgArg-NH 2 (compound 1).
- Lys, Ser, Pro, Arg, His, and GIn are standard 3-letter codes for lysine, serine, proline, arginine, histidine, and glutamine.
- Ser(PO 4 2" ) represents phosphoserine.
- FIG. 6 illustrates an exemplary kinase substrate comprising two recognition moieties that can be used to detect a protein kinase that recognizes the peptide consensus sequence for protein kinase p38/3II, i.e., Cl l-OO-Lys-(Dye 2)-ArgArgIleProLeuSerProLsySerPro-OO- LyS-(Cn)-NH 2 (compound 2).
- the two recognition moieties are highlighted in bold.
- Dye 2 is 5-carboxy-2',7'-dipyridyl-sulfonefluorescein and tet is 2',7',4,7-tetachloro-5-carboxy fluorescein.
- Arg, He, Pro, Leu, Ser, Thr, and Lys are standard 3-letter codes for arginine, isoleucine, proline, leucine, serine, threonine, and lysine.
- An exemplary synthesis for compound 2 is described in Example 1.
- compound 2 contains a sulfonate anion in the Dye moiety, for a total negative charge of -2. This is offset by the guanidinium groups in the two arginine residues, for a total of two positive charges. Thus, the net charge of the compound is about 0 at pH 7.6.
- Compound 2 further includes two protein kinase recognition moieties comprising two unphosphorylated serines in the form of a polypeptide containing the amino acid sequence, ProLeuSerProLsySerPro, that is recognized by protein kinase p38/3II.
- protein kinase recognition moieties comprising two unphosphorylated serines in the form of a polypeptide containing the amino acid sequence, ProLeuSerProLsySerPro, that is recognized by protein kinase p38/3II.
- a comparison of the rates of reaction for a kinase substrate comprising two protein kinase recognition moieties and two hydrophobic moieties i.e. CnOOK(dye 2)RRIPLSPLSPOOK(C 1 ONH 2 , used at a concentration of 8 ⁇ m and referred to herein as compound 2) versus a kinase substrate comprising a single protein kinase recognition moiety and two hydrophobic moieties ⁇ i.e., CnOOK(dye 2)RRIPLSP00K(Cii)NH 2 ), used at a concentration of 8 ⁇ m) for two concentrations of ATP (10 and 100 ⁇ M) is shown in FIG. 1OA and 1OB.
- R, I, P, L, S, and K are standard 1 -letter codes for arginine, isoleucine, proline, leucine, serine and lysine.
- the rates of the reaction were fitted to the Michaelis-Menton equation.
- the protein kinase substrate with two protein kinase recognition moieties provided improved signal to background ratios. As shown in FIG.
- the kinase substrate C ⁇ OOK(dye 2)RRtPLSPLSPOOK(Cn)NH 2 (referred to herein as compound 2) comprising two protein kinase recognition moieties has an improved signal to background ratio as compared to the kinase substrate comprising one protein kinase recognition moiety, C n OOK(dye 2)RRIPLSP00K(Ci,)NH 2 .
- Micelle formation can be particularly favored when the charge on the substrate molecule is balanced by the charge on the charge-balance moiety(ies) so that the net charge is approximately zero, or a small negative or small positive net charge, so that micelle formation is not prevented by mutual charge repulsion. While not intending to be bound by any theory of operation, it is believed that ionic bonds can be formed between oppositely charged charge-balance moiety(ies) and any other moieties described herein in aqueous solution at physiological pH and promote or encourage micelle formation. For example, FIG.
- C i 6 RROOORRIYGRF quenches the fluorescence of a substrate molecule
- FIG. 12 illustrates the rate of the reaction for a tyrosine kinase using 2 ⁇ M substrate molecule (Ci 6 Lys(Dye 2)OOOGluGluIleTyrGlyGluPheNH 2 ) and 2 ⁇ M charge-balance molecule (Ci 6 ArgArgOOOArgArgIleTyrGlyArgPheNH 2 ), and 0 or 100 ⁇ M ATP, and 5 nM tyrosine kinase Lyn.
- the addition of tyrosine kinase Lyn to the micelle comprising the substrate molecule and charge-balance molecule cause an increase in fluorescence over time.
- the present disclosure contemplates not only detecting enzymes, but also methods involving: (1) screening for and/or quantifying enzyme activity in a sample, (2) determining kcat and/or Km of an enzyme or enzyme mixture with respect to selected substrates, (3) detecting, screening for, and/or characterizing substrates of enzymes, (4) detecting, screening for, and/or characterizing inhibitors, activators, and/or modulators of enzyme activity, and (5) determining substrate specificities and/or substrate consensus sequences or substrate consensus structures for selected enzymes.
- a sample that contains, or can contain, a particular enzyme activity is mixed with a substrate of the present teachings, and the fluorescence is measured to determine whether an increase in fluorescence has occurred. Screening can be performed on numerous samples simultaneously in a multi-well or multi- reaction plate or device to increase the rate of throughput.
- Kcat and Km can be determined by standard methods, as described, for example, in Fersht, Enzyme Structure and Mechanism, 2nd Edition, W.H. Freeman and Co., New York, (1985)).
- the reaction mixture can contain two or more different enzymes. This can be useful, for example, to screen multiple enzymes simultaneously to determine if an enzyme has a particular enzyme activity.
- the substrate specificity of an enzyme can be determined by reacting an enzyme with different substrate molecules having different substrate moieties the activity of the enzyme toward the substrates can be determined based on an increase in fluorescence. For example, by reacting a protein kinase with several different substrate molecules having several different protein kinase recognition moieties, a consensus sequence for preferred substrates of the kinase can be determined.
- each different substrate may be tested separately in different reaction mixtures, or two or more substrates may be present simultaneously in a reaction mixture.
- the substrates can contain the same fluorescent moiety, in which case the observed fluorescent signal is the sum of the signals from enzyme reaction with both substrates.
- the different substrates can contain different, fluorescently distinguishable fluorescent moieties that allow separate monitoring and/or detection of the reaction of enzyme with each different substrate simultaneously in the same mixture.
- the fluorescent moieties can be selected such that all or a subset of them are excitable by the same excitation source, or they may be excitable by different excitation sources. They can also be selected to have additional properties, such as, for example, the ability to quench one another when in close proximity thereto, by, for example, collisional quenching, FRET or another mechanism (or combination of mechanisms).
- the assay mixture may optionally include one or more amphipathic quenching compounds designed to quench the fluorescence of the fluorescent moiety of the substrate (and/or plurality of substrates when more than one substrate is present in the mixture).
- amphipathic quenching molecules generally comprise a hydrophobic moiety capable of integrating the quenching compound into a micelle and a quenching moiety.
- the hydrophobic moiety can by any moiety capable of integrating the compound into a micelle, and as specific nonlimiting exemplary embodiments, can comprise any of the hydrophobic moieties described previously in connection with, for example, the kinase substrates.
- the quenching moiety can include any moiety capable of quenching the fluorescence of the fluorescent moiety of the enzyme substrate used in the assay (or one or more of the substrates if a plurality of substrates are used).
- Compounds capable of quenching the fluorescence of the various different types of fluorescent dyes discussed above, such as xanthene, fluorescein, rhodamine, cyanine, phthalocyanine and squaraine dyes are well- known.
- Such quenching compounds can be non-fluorescent (also referred to as "dark quenchers" or "black hole quenchers") or, alternatively, they may themselves be fluorescent.
- suitable fluorescent quenchers include, but are not limited to, the various fluorescent dyes described above in connection with kinase substrates.
- the ability of a quencher to quench the fluorescence of a particular fluorescent moiety may depend upon a variety of different factors, such as the mechanisms of action by which the quenching occurs.
- the mechanism of the quenching is not critical to success, and may occur, for example, by collision, by FRET, by another mechanisms or combination of mechanisms.
- the selection of a quencher for a particular application can be readily determined empirically.
- the dark quencher Dabcyl and the fluorescent quencher TAMRA have been shown to effectively quench the fluorescence of a variety of different fluorophores.
- a quencher can be selected based upon its spectral overlap properties spectral overlap with the fluorescent moiety.
- a quencher can be selected that has an absorbance spectrum that sufficiently overlaps the emission spectrum of the fluorescent moiety such that the quencher quenches the fluorescence of the fluorescent moiety are in close proximity to one another, such as when the quencher molecule and substrate including the quencher moiety are integrated into the same micelle.
- hydrophobic and quenching moieties can be connected in any way that permits them to perform their respective functions.
- only one of the two hydrophobic moieties may be linked either directly or via a linker to a quenching moiety.
- both hydrophobic moieties may be linked either directly or via a linker to a quenching moiety.
- one hydrophobic moiety may be linked directly to the quenching moiety without the aid of a linker.
- Non-limiting examples of such quenching compounds include molecules in which a dye (e.g. a rhodamine or fluorescein dye) which contains a primary amino group (or other suitable group) is acylated with a fatty acid.
- the linkage may be mediated by way of a linker.
- the identity of the linker is not critical, and can include a peptide segment (or analog thereof).
- the peptide segment will not include an enzyme recognition moiety recognized by the enzyme(s) being assayed, it may optionally include such a . moiety(ies).
- the quencher molecule can be a derivative or analog of any of the kinase or other enzyme substrates described herein in which the fluorescent moiety is replaced with a quenching moiety and the sequence of the enzyme recognition moiety is modified such that it is not recognized by the enzyme(s) being assayed in the sample.
- the quencher molecule can be designed to have specified charge characteristics.
- Detecting, screening for, and/or characterizing inhibitors, activators, and/or modulators of enzyme activity can be performed by forming reaction mixtures containing such known or potential inhibitors, activators, and/or modulators and determining the extent of increase or decrease (if any) in fluorescence signal relative to the signal that is observed without the inhibitor, activator, or modulator. Different amounts of these substances can be tested to determine parameters such as Ki (inhibition constant), K H (Hill coefficient), Kd (dissociation constant) and the like to characterize the concentration dependence of the effect that such substances have on enzyme activity.
- Detection of fluorescent signal can be performed in any appropriate way.
- substrate molecules/charge-balance molecules of the present teachings can be used in a continuous monitoring phase, in real time, to allow the user to rapidly determine whether enzyme activity is present in the sample, and optionally, the amount or specific activity of the enzyme.
- the fluorescent signal is measured from at least two different time points, usually until an initial velocity (rate) can be determined.
- the signal can be monitored continuously or at several selected time points.
- the fluorescent signal can be measured in an end-point embodiment in which a signal is measured after a certain amount of time, and the signal is compared against a control signal (before start of the reaction), threshold signal, or standard curve. 6.8 Kits
- kits for performing methods of the present teachings generally comprise one or more molecules that collectivity include three to four different types of moieties: a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety. Any of the various compositions described above can be used in the kits.
- the kit comprises a substrate molecule comprising one or more hydrophobic moieties, one or more fluorescent moieties, a substrate moiety comprising two or more recognition moieties for a target enzyme, and a buffer for preparing a reaction mixture that facilitates the enzyme reaction.
- the kit comprises a substrate molecule comprising one or more hydrophobic moieties, one or ore fluorescent moieties, a substrate moiety comprising one or more enzyme recognition moieties, a charge-balance moiety, and a buffer for preparing a reaction mixture that facilitates the enzyme reaction.
- the kit comprises a substrate molecule and a charge balance molecule.
- the substrate molecule comprises one or more hydrophoic moieties and a substrate moiety.
- the charge-balance molecule comprises at least one hydrophobic moiety and a charge-balance moiety.
- One, or both, of the substate and/or charge-balance molecules can further comprise a fluorescent moiety.
- Kits comprising additional moieties and/or molecules are also envisaged.
- a quenching moiety can be included in the substrate molecule, the charge-balance molecule, in both the substrate molecule and the charge-balance molecule, or in a distinct quenching molecule.
- the buffer can be provided in a container in dry form or liquid form.
- the choice of a particular buffer can depend on various factors, such as the pH optimum for the enzyme to be detected, the solubility and fluorescence properties of the fluorescent moiety in the substrate molecule and/or charge-balance molecule, and the pH of the sample from which the target enzyme is obtained.
- the buffer is usually added to the reaction mixture in an amount sufficient to produce a particular pH in the mixture.
- the buffer is provided as a stock solution having a pre-selected pH and buffer concentration. Upon mixture with the sample, the buffer produces a final pH that is suitable for the enzyme assay, as discussed above.
- the pH of the reaction mixture can also be titrated with acid or base to reach a final, desired pH.
- the kit can additionally include other components that are beneficial to enzyme activity, such as salts (e.g., KCl, NaCl, or NaOAc), metal salts (e.g., Ca2+ salts such as CaCl 2 , MgCl 2 , MnCl 2 , ZnCl 2 , or Zn(OAc), detergents (e.g., TWEEN 20), and/or other components that can be useful for a particular enzyme.
- salts e.g., KCl, NaCl, or NaOAc
- metal salts e.g., Ca2+ salts such as CaCl 2 , MgCl 2 , MnCl 2 , ZnCl 2 , or Zn(OAc
- detergents e.g., TWEEN 20
- the molecules that collectivity include four different types of moieties: a hydrophobic moiety, a fluorescent moiety, a substrate moiety and a charge-balance moiety can be provided in dry or liquid form, together with or separate from the buffer.
- the substrate molecule and/or charge-balance molecule can be provided in an aqueous solution, partially aqueous solution, or non-aqueous stock solution that is miscible with the other components of the reaction mixture.
- a substrate solution can also contain a cosolvent such as dimethyl formamide, dimethylsulfonate, methanol or ethanol, typically in a range of l%-10% (v:v).
- the kit can also contain additional chemicals useful in the detection, quantifying, and/or characterizing of enzymes.
- additional chemicals useful in the detection, quantifying, and/or characterizing of enzymes.
- the kit can also contain a phosphate-donating group, such as ATP, GTP, ITP (inosine triphosphate) or other nucleotide triphosphate or nucleotide triphosphate analog that can be used by the kinase to phosphorylate the substrate moiety.
- a phosphate-donating group such as ATP, GTP, ITP (inosine triphosphate) or other nucleotide triphosphate or nucleotide triphosphate analog that can be used by the kinase to phosphorylate the substrate moiety.
- Example 1 Preparation of Protein Kinase Substrates [0223] Resins and reagents for peptide synthesis, Fmoc amino acids, 5-carboxyfluorescein succinimidyl ester were obtained from Applied Biosystems (Foster City, CA). Fmoc- Lys(Mtt)-OH, Fmoc-Ser(OPO(OBzl(OH)-OH and Fmoc-Dpr(ivDde) were obtained from Novabiochem. All other chemicals and buffers were obtained from Sigma/ Aldrich.
- Peptide synthesis was performed on an Applied Biosystems Model 433 A Peptide Synthesizer.
- HPLC was performed on an Agilent 1100 series HPLC.
- UV- Vis measurements were performed on a Cary 3E UV- Vis spectrophotometer.
- MALDI Mass spectral data were obtained on an Applied Biosystems Voyager using cyano-4-hydroxycinnamic acid as matrix material.
- An exemplary enzyme substrate useful for detecting protein kinase p38/3II, C 11 - OOK(dye2)RRIPLSPLSPOOK(C ⁇ )-amide was prepared as follows.
- the peptide OOK(ivDde)RRIPLSPLSPOOK(Mtt) was constructed via solid phase peptide synthesis using standard FastMocTM chemistry on 125 mg of Fmoc-P AL-PEG-PS resin at 0.16 mmol/g, a solid support which results in a carboxamide peptide.
- the resin was washed with DMF (5x1 mL) and treated with 10% hydrazine in DMF for ten minutes.
- 5-Carboxy- 2 ' ,7'-dipyridylsulfonefluorescein ( 10 mg), HBTU/HOBT solution (0.1 mL) and diisopropylethylamine (0.04 mL) were added to the resin and the mixture agitated for 45 minutes.
- the resin was washed with 8x1 mL DMF, 1x1 mL acetonitrile.
- the peptide was cleaved from the resin with 1 mL cleavage solution (950 ⁇ L TFA, 50 ⁇ L water).
- An exemplary substrate molecule useful for detecting protein tyrosine kinase Lyn Ci 6 Lys(Dye 2)OOOGluGluIleTyrGlyGluPheNH2 was prepared as follows.
- the peptide OOOK(ivDde)GluGluIleTyrGlyGluPhe(Mtt) was constructed via solid phase peptide synthesis using standard FastMocTM chemistry on 125 mg of Fmoc-P AL-PEG-P S resin at 0.16 mmol/g, a solid support which results in a carboxamide peptide.
- the resin was washed with DMF (5x1 mL) and treated with 10% hydrazine in DMF for ten minutes.
- 5-Carboxy- 2',7'-dipyridylsulfonefluorescein (10 mg), HBTU/HOBT solution (0.1 mL) and diisopropylethylamine (0.04 mL) were added to the resin and the mixture agitated for 45 minutes.
- the resin was washed with 8x1 mL DMF, 1x1 mL acetonitrile.
- the peptide was cleaved from the resin with 1 mL cleavage solution (950 ⁇ L TFA, 50 ⁇ L water).
- Ci 6 ArgArgOOOArgArgIleTyrGlyArg PheNH 2 useful for balancing the charge of substrate molecule Ci 6 Lys(Dye 2)OOOGluGluIleTyrGlyGluPheNH 2
- the peptide ArgArgOOOArgArgIleTyrGlyArgPheNH2 (Mtt) was constructed via solid phase peptide synthesis using standard FastMocTM chemistry on 125 mg of Fmoc-P AL-P EG-PS resin at 0.16 mmol/g, a solid support which results in a carboxamide peptide.
- the resin was washed with DMF (5x1 mL) and treated with 10% hydrazine in DMF for ten minutes.
- 5-Carboxy- 2',7'-dipyridylsulfonefluorescein (10 mg), HBTU/HOBT solution (0.1 mL) and diisopropylethylamine (0.04 mL) were added to the resin and the mixture agitated for 45 minutes.
- the resin was washed with 8x1 mL DMF, 1x1 mL acetonitrile.
- the peptide was cleaved from the resin with 1 mL cleavage solution (950 ⁇ L TFA, 50 ⁇ L water).
- Example 3 Addition of Charge-Balance Molecule Quenches the Fluorescence of the Substrate Molecule
- a reaction solution was prepared containing 10 ⁇ M substrate molecule C16Lys(Dye 2)OOOGluGluIleTyrGlyGluPheNH2 and 25 niM Tris (pH 7.6), 5 mM MgCl and 5 niM DTT. Varying concentrations of the charge-balance molecule Ci 6 ArgArgOOOArgArgIleTyrGlyArg PheNH 2 were added (final concentration 0, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 50 ⁇ M) and the fluorescence was determined. The results are shown in Fig. 11.
- Example 4 Detection of Protein Kinase Activity
- Kinase assays were performed using Corning 384- well, black, non-binding surface (NBS), microwell plates. Fluorescence was read in real time using a Molecular Dynamics Gemini XS plate reader, with excitation and emission set at 500 and 550 respectively. The plate was read every minute for one hour at ambient temperature.
- a reaction solution was prepared containing the substrate molecule C 16 Lys(Dye 2)OOOGluGluIleTyrGlyGluPheNH 2 (2 ⁇ M), and charge-balance molecule Ci 6 ArgArgOOOArgArgIleTyrGlyArg PheNH2 (2 ⁇ M), 20 mM Tris buffer, pH 7.6, MgCl 2 (5 mM), DTT (5 mM) and Lyn (5 nM).
- the solution was pipetted into wells of a 384-well plate (10 mL per well). ATP (0 or 100 ⁇ M ) was added to initiate the kinase reaction. The plate was read at 500 nm excitation, 550 nm emission, each minute for 1 hour. The results are shown in Fig. 12.
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