Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

• protein kinases represent one of the largest superfamilies of enzymes in eukaryotic organisms, with an estimated 1-3% of the human genome coding for various protein kinases. Protein kinases catalyze the transfer of phosphate from
• ATP to specific amino acids in proteins, and phosphorylation of proteins is known to be the most widespread mechanism for reversible covalent modification of protein structure and function.
• the dysfunctional regulation of protein phosphorylation is believed to result in several diseases, such as, for example, diabetes, cancer, and many forms of heart disease.
• the ability to assay the activity of the various intracellular processes responsible for the covalent modification of particular biomolecules is essential in order to gain an understanding of the potential roles such processes play in normal cells and various disease states.
• Assay techniques which detect and quantify various types of covalent modifications of particular biomolecules would also facilitate the development of diagnostic and therapeutic technologies relating to disease states resulting from dysfunctional modification processes.
• An assay technique ideally suited for these purposes would be sensitive and continuous, would allow both in vitro and in vivo assays, would be efficient and economical, and would enable high-density, high- throughput screening.
• the assay technique disclosed in Geoghegan et al. requires the interaction of two expensive components (i.e., the interaction of double-labeled tyrosine kinase substrate peptides with SH2 domain peptides), resulting in a technique that is relatively complex and expensive. Moreover, due to its use of a multi-component system, the assay technique of Geoghegan et al. is not suitable for performing in vivo assays. Homogenous fluorescent protein kinase assay methods utilizing various Green
• GFP Fluorescent Protein
• Nagai et al. teaches a method for assaying protein kinase activity using a Kinase-Inducible Domain construct containing two GFP groups (See, Nagai et al., Nature Biotechnology, 18:313-316 (2000)).
• the assay method taught by Nagai et al. depends upon phosphorylation-dependent changes in the fluorescence resonance energy transfer ("FRET") among the two GFP groups to detect kinase activity, and because such changes in FRET are small, the assay technique of Nagai et al. does not provide a homogenous assay having a desired level of sensitivity.
• FRET fluorescence resonance energy transfer
• Patent 5,912,137 (“the ' 137 Patent”) teaches a protein kinase assay utilizing modified GFP molecules as assay substrates.
• the assays taught in the ' 137 patent can only be carried out using modified GFP substrates, the potential applications of such assays are limited.
• the present invention includes substrates and methods useful for the assay of covalent modification of biomolecules.
• the assay methods of the present invention are sensitive and homogeneous and do not require the use of radioisotopes.
• the assay methods herein disclosed are also relatively simple and economical, adaptable to a wide variety of applications, easily used in vitro and in living cells, and allow continuous, real-time monitoring of structural modifications to biomolecules.
• the assay methods of the present invention are useful for detecting and quantifying a wide range of covalent biomolecular modifications which do not result in the cleavage of the biomolecule.
• the present invention offers significant advantages in terms of simplicity, efficiency, and scope when compared to presently used methods for detecting covalent biomolecular modifications.
• the substrates of the present invention are double-labeled biomolecular substrates (the phrases "double-labeled biomolecular substrate” and “double-labeled substrate” are used interchangeably herein).
• the double-labeled substrates of the present invention include a core molecular backbone covalently labeled at two positions with a first fluorescent dye and a second fluorescent dye or with a first fluorescent dye and a second non-fluorescent dye (for convenience, the term “dye” is used herein to describe a chromophoric or fluorophoric moiety).
• the core molecular backbone of a double-labeled substrate according to the present invention may include a protein or peptide sequence, a nucleotide sequence, a sugar, a lipid, a receptor, or a biopolymer.
• biopolymer includes any molecule that is a covalent combination of amino acids, nucleic acids, sugars, lipids, or other small molecules of biological origin.
• the core molecular backbone may also include a substrate determinant specific for a particular process of covalent biomolecular modification.
• the invention enables homogenous and continuous assay methods which are simple and economical, and which may be employed both in vitro and in living cells.
• the quenching phenomenon underlying the substrates and fluorescence assay methods of the present invention is not fluorescence resonance energy transfer ("FRET"). Unlike FRET, the quenching phenomenon underlying the substrates and fluorescence assay methods of the present invention involve ground state interaction of two dyes.
• double-labeled substrates of the present invention may be used for homogenous absorbance-based assays detecting various types of structural modifications of biomolecules through modification-dependent changes in the absorbance spectra of the double-labeled substrates.
• the assay methods of the present invention are versatile. Because the core molecular backbone of double-labeled substrates of the present invention can be constructed to include substrate determinants for a wide range of intracellular processes, the assay methods of the present invention are applicable to a broad range of covalent biomolecular modifications.
• the core molecular backbone of a double-labeled substrate may include a protein kinase substrate determinant.
• Such a double-labeled substrate could then be used to assay the activity of a particular protein kinase or a particular class of protein kinases. However, this is but one example.
• FIG. 3 schematically depicts the modification-dependent transition between intramolecular dimer and intramolecular monomer states of a double-labeled biomolecular substrate according to the present invention.
• FIG. 4 is a schematic illustration of a second embodiment of a double-labeled biomolecular substrate of the present invention in a structurally modified state.
• FIG. 5 illustrates the absorbance spectrum of double-labeled substrate including a fluorescein and a rhodamine label before and after phosphorylation by PKA.
• FIG. 6 illustrates the fluorescent emission of the fluorescein label of the double- labeled substrate of FIG. 5 before and after phosphorylation by PKA.
• FIG. 7 illustrates the fluorescent emission of the rhodamine label of the double- labeled substrate of FIG. 5 before and after phosphorylation by PKA.
• FIG. 10 schematically illustrates a double-labeled substrate including a covalently attached enhancer, the double-labeled substrate transitioning between an unphosphorylated and a phosphorylated state upon phosphorylation by PKA.
• FIG. 2 provides a schematic illustration of the double-labeled substrate 10 of FIG. 1 after the double-labeled substrate 10 has been structurally modified by, for example, a protein kinase.
• the protein kinase adds a phosphate group 60 to an amino acid residue of the substrate determinant 30 included in the core molecular backbone 20 of the double-labeled substrate 10 (protein kinases most commonly phosphorylate hydroxyl amino acids, such as serine, threonine, and tyrosine).
• Phosphorylation of the double-labeled substrate 10 results in the dissociation of the fluorescent dye 40 and the quenching dye 50 and, thus, the dissociation of the intramolecular dye dimer 55, which, in turn, results in a marked increase in the fluorescence of the double-labeled substrate because the quenching dye 50 no longer quenches the fluorescence of the fluorescent dye 40.
• the double-labeled substrate 10 only exhibits a change in fluorescence upon covalent modification, in this case phosphorylation.
• the extent to which an amount of double-labeled substrate of the present invention is structurally modified can be continually assayed without separation steps merely by quantifying various changes in substrate fluorescence.
• FIG. 3 schematically illustrates the modification dependent transition between intramolecular dimer and intramolecular monomer of the two dyes 80, 90 of a double- labeled substrate 70 of the present invention using a peptide based on the Kinase- Inducible Domain (KID) of the Cyclic AMP Response Element Binding Protein (CREB) and Protein Kinase A (“PKA”) as an illustrative system.
• KID Kinase- Inducible Domain
• CREB Cyclic AMP Response Element Binding Protein
• PKA Protein Kinase A
• FIG. 3 illustrates a double- labeled substrate 70 of the present invention which includes the KID sequence of CREB within the core molecular backbone 75, and two dyes 80, 90 conjugated to the KID sequence.
• the two dyes 80, 90 form an intramolecular dye dimer 95 and the fluorescence of the fluorescent dye 80 is quenched.
• a phosphate group, or "P" 100 is introduced in the double-labeled substrate 70, and the intramolecular dye dimer 95 dissociates, resulting in an increase in the fluorescence of the flourescent dye 80.
• the double- labeled substrates of the present invention are useful for assaying a broad range of structural modifications to various biomolecules and can be specifically constructed for the assay of numerous processes of covalent biomolecular modification.
• the core molecular backbone of double-labeled substrates of the present invention may be constructed to include protein or peptide sequences, nucleotide sequences, sugars, lipids, receptor molecules, biopolymers, or virtually any other biomolecule which may serve as a substrate in one or more intracellular processes of covalent biomolecular modification.
• double-labeled substrates of the present invention can be constructed for the assay of numerous catalytic and non-catalytic processes of covalent biomolecular modification.
• double-labeled substrates according to the present invention could be constructed to exhibit a change in fluorescence upon sulfation, glycation, glycosylation, carboxylation, myristoylation, farnesylation, ubiquitination, biotinylation, or other modification reactions.
• the following explanations might explain the excellent results of the invention.
• the dissociation of intramolecular dye dimer formed by the two dyes could be the result of one or more different mechanisms.
• the enhancer molecule might be a phosphotyrosine-specific antibody or a SH2-domain-containing protein with a high affinity for phosphotyrosine.
• an enhancer molecule 200 could be covalently combined with the double-labeled substrate 10 to form a single chimeric molecule.
• a second embodiment of the double-labeled substrate of the present invention is schematically illustrated in FIG. 4.
• the double-labeled substrate 110 of FIG. 4 is illustrated in its modified state for ease of description and includes a core molecular backbone 120, a substrate determinant 125 within the core molecular backbone 120, a first spacer segment 130 and a second spacer segment 140.
• the first spacer segment 130 is included at a first terminus 135 of the core molecular backbone 120
• the second spacer segment 140 is included at a second terminus 145 of the core molecular backbone 120.
• the first or second spacer segments 130, 140 may also be included in the double-labeled substrate 110 in order to facilitate the use of a particular dye which cannot be conjugated to one of the amino acid residues included in the core peptide sequence 120.
• the embodiment illustrated in FIG. 4 highlights that the exact construction of double- labeled biomolecular substrates of this invention will vary.
• the construction of a double-labeled substrate may vary not only by including one or more spacer segments which may facilitate a more sensitive assay or enable the use of different dyes, but the construction may vary to utilize biomolecular substrates corresponding to different processes of covalent biomolecular modification.
• double-labeled substrates of the present invention could be constructed using core molecular backbones which include, among others, the following amino acid sequences: Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser (SEQ. ID. NO.: 9), a substrate peptide for Protein Kinase A and Protein Kinase C (See, e.g., PCT International Application WO Patent Document 98/09169); Phe-Arg-Arg-Leu-Ser-Ile- Ser-Thr (SEQ. ID. NO.: 1) and Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser (SEQ. ID.
• the first or second spacer segments 130, 140 may also be included in the double-labeled substrate 110 in order to facilitate the use of a particular dye which cannot be conjugated to one of the amino acid residues included in the core peptide sequence 120.
• the embodiment illustrated in FIG. 4 highlights that the exact construction of double- labeled biomolecular substrates of this invention will vary.
• the construction of a double-labeled substrate may vary not only by including one or more spacer segments which may facilitate a more sensitive assay or enable the use of different dyes, but the construction may vary to utilize biomolecular substrates corresponding to different processes of covalent biomolecular modification.
• double-labeled substrates of the present invention illustrate only a few of the potential substrate determinants which may be included in double-labeled substrates of the present invention. These examples do not reflect the myriad of other -11- biomolecular substrates which may be included in the core molecular backbone of double-labeled substrates of the present invention.
• the double-labeled substrates of the present invention have broad application and can be tailor-made for use with any one of many enzymatically catalyzed or non-catalyzed intracellular processes by which biomolecules are covalently modified.
• a double-labeled substrate according to the present invention may include a non-fluorescent dye and a fluorescent dye, or, alternatively, a double labeled substrate according to the present invention may be constructed using two fluorescent dyes.
• a preferred method for assaying structural modification of biomolecules in vitro is homogenous, comparatively simple, and includes the steps of providing a double-labeled substrate as herein described, including the double-labeled substrate in a sample, and quantifying any resultant change in fluorescence or absorbance resulting from the structural modification of the double- labeled substrate. Because only double-labeled substrate which is structurally modified exhibits a change in fluorescence or absorbance, this method requires no separation of the unmodified double-labeled substrate from the modified double-labeled substrate before the modification of the double-labeled substrate can be accurately assayed.
• the assay methods of the present invention require no special reagents other than the double-labeled substrate, and the measurement of changes in fluorescence or absorbance of the double-labeled substrate can be easily achieved using a variety of well known instruments, such as, for example, known spectrometers, 96-well and 384- -13- well microtiter plate readers, other multichannel readers, and micro-array instruments. Therefore, the methods of assaying covalent biomolecular modifications according to the present invention provide advantages over currently used assays in terms of simplicity, throughput, versatility, and economy. Because the method already described requires no separation steps, it can be easily modified in order to assay processes of covalent biomolecular modification in living cells.
• the double-labeled substrates of the present invention can also be used in methods facilitating the discovery of drugs which target intracellular processes of covalent biomolecular modification.
• Such a preferred method would include the steps of providing a sample containing the modifying enzyme(s) to be targeted, introducing into the sample a drug designed to target a particular intracellular process of covalent biomolecular modification, introducing into the sample a double-labeled substrate specific for the targeted modification process, and quantifying any change in fluorescence or absorbance resulting from the structural modification of the double- labeled substrate using well known instruments.
• kits which utilize the double-labeled substrates and methods described herein to detect and/or quantify covalent biomolecular modification.
• a preferred embodiment of such a kit would include a container, one or more different double-labeled substrates of the present invention contained within the container, and instructions for use.
• the kits may also include, for convenience, buffers and other reagents necessary to carry out the assay, and samples of enzyme for calibration purposes.
• the reagents included with the kits can be varied depending on the application and in order to optimize the sensitivity of the assay.
• the protein kinase activity would result in a covalent structural modification of the double-labeled substrate, leading to a change in fluorescence or absorbance and in situ detection of kinase activity using instruments well known in the art, such as, for example, known spectrometers, fluorescence microscopes, plate readers, cell counters, and cell sorters.
• Example I A double-labeled protein kinase substrate can be designed, synthesized, characterized, and used to assay the activity of PKA and other protein kinases.
• the Lys residue of the native KID sequence was replaced by an Arg residue to facilitate site specific labeling of the peptide's ⁇ -amino group.
• Replacing the Lys residue of the native KID sequence resulted in the synthetic KID sequence Arg-Arg- Pro-Ser-Tyr-Arg-Arg-Ile-Leu-Asn- Asp-Leu (SEQ. ID.
• the KID sequence represents the sequence generally referred to herein as "the KID sequence.”
• a Cys residue was added to the C-terminus of the synthetic KID sequence to allow labeling of the molecular backbone with a dye through the sulfhydryl group in the cysteine residue, and a Gly residue was added at the terminal Cys residue to facilitate peptide synthesis.
• the additional peptide sequence Asp-Ser-Gln-Arg-Arg-Arg-Glu-Ile-Leu-Ser (SEQ. ID. NO.: 8) was added at the Arg residue of the N-terminus of the KID sequence to give the final peptide more helical structure.
• the synthetic peptide sequence of the core molecular backbone of the substrate was synthesized on a benzhydrylamine resin using conventional (tBOC) solid phase peptide synthetic chemistry. See, e.g. Barany and Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, eds., (Acad. Press, New York, 1980), Glass. D.B., Methods Enzymol., 99, 119-139 (1983).
• the core molecular backbone was conjugated with two dyes to form a double-labeled substrate.
• the synthetic peptide sequence of the core molecular backbone was conjugated with tetramethylrhodamine-5-maleimide.
• the maleimide on the dye reacts with the cysteine residue at the C-terminus in the KID region, and the maleimide group serves as the link between the sulfhydryl group on the cysteine and the rhodamine group.
• the single-labeled substrate was then conjugated at the N-terminus with either 5-carboxyfluorescein, succinimidyl ester or 5- carboxytetramethylrhodamine, succinimidyl ester.
• succinimidyl ester group reacts with the amino group at the N-terminus of each peptide to form a carboxamide bond with the dye.
• HPLC purification on a C4 reversed-phase column the double-labeled substrate was subjected to analysis by mass spectrometry analysis, UV absorbance spectrophotometry, and fluorescence spectrophotometry. After such analyses, the double-labeled substrate was phosphorylated with PKA.
• the phosphate acceptor amino acid in this double-labeled substrate is the serine residue found within the embedded KID sequence.
• FIG. 6 and 7 illustrate the even more dramatic phosphorylation dependent changes in the fluorescence characteristics a double-labeled substrate having a fluorescein and a rhodamine molecule conjugated thereto.
• the fluorescence of the fluorescein label increased 340% after phosphorylation
• FIG. 7 illustrates that phosphorylation of the double-labeled substrate caused a 35% increase in the fluorescence of the rhodamine label.
• the molecular backbone described herein may also be labeled with, among other combinations, two rhodamine dyes instead of a rhodamine dye and a fluorescein dye, and FIG. 8 and FIG. 9 illustrate the phosphorylation-dependent changes in the optical properties of such a double-labeled substrate.
• the double-labeled substrate in its unphosphorylated state, the double-labeled substrate exhibited two absorbance maxima. The larger peak is at 520 nm, while the smaller peak is at 552 nm. After phosphorylation, the peak at 520 nm decreases in size while the peak at 552 nm shifts to 550 nm and increases in size.
• FIG. 8 and FIG. 9 illustrate the phosphorylation-dependent changes in the optical properties of such a double-labeled substrate.
• FIG. 8 and FIG. 9 illustrate the phosphorylation-dependent changes in the optical properties of such a double-labeled substrate.
• the double-labeled substrate in its
• phosphorylation of the double-labeled substrates results in an increase in the intensity of the fluorescent emission peak of at least one dye conjugated to the double-labeled substrates.
• Sensitivity is also excellent with changes in dye emission intensity being observable at low nanomolar concentrations of peptide in a standard spectrofluorometer.
• the favorable sensitivity and signal-to-noise ratio indicate the double-labeled substrate will be useful for monitoring protein kinase activity in a variety of applications.
• the procedures and methods described herein can be employed to prepare and use double-labeled protein kinase substrates for assaying most any other protein kinase.
• the KID sequence included in the core molecular backbone described -19- herein may be modified to contain an appropriate consensus sequence for a given protein kinase determinant.
• consensus sequences can be found in the literature for many common kinases such as PKA, PKC, CaM kinase II, etc. (cf, Songyang et al. Current Biol. 4:479. 1994).
• the double-labeled substrate of the present invention can be prepared for assaying most any other intracellular processes leading to the structural modification of protein or other biomolecules.

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