Patent Description:
The present disclosure generally relates to assays of enzymes responsible for phosphoregulation including kinases (such as protein kinases, which mediate protein and peptide phosphorylation) and phosphatases (such as protein phosphatases, which mediate protein and peptide dephosphorylation). For clarity, the descriptions provided generally focus on protein kinases to illustrate certain astpects of this disclosure, but it should be understood that in other aspects, phosphatases may be used.

Protein kinases are involved in aspects of regulation within cells. Protein kinases generally act by adding phosphoryl groups (phosphorylation) to certain amino acid residues such as serine, threonine, or tyrosine. Phosphorylation often results in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins. Protein kinases are found in a wide variety of organisms, including animals, plants, and bacteria. Detection of phosphorylation events within proteins is often done with fluorescence, but can be difficult due to background fluorescence, e.g., in the presence of large libraries of small molecule organic compounds. Accordingly, improvements are needed.

<NPL>, relates to interrogating endogenous protein phosphatase activity with rationally designed chemosensors. <NPL>, relates to a luminescent sensor for tyrosine phosphorylation. <NPL>, relates to fluorescent reporters and biosensors for probing the dynamic behavior of protein kinases.

The present disclosure generally relates to assays of kinases such as protein kinases, which mediate protein and peptide phosphorylation. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present disclosure is generally directed to a composition as defined in the claims. In one set of aspects, the composition comprises a peptide having a first portion comprising a structure:
<CHM>
where each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently is hydrogen or -SO<NUM>X such that at least one of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is -SO<NUM>X, where X is -OR' or -NR'R", R' and R" each independently being hydrogen or an alkyl, and n is <NUM> or a positive integer, and a second portion comprising a phosphate group, where the N and/or the O of the first portion, and the phosphate group of the second portion, are coordinated via a europium ion.

The composition, in another set of aspects, comprises a solution comprising dissolved europium ions and a peptide comprising a portion having a structure:
<CHM>
where each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently is hydrogen or -SO<NUM>X such that at least one of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is -SO<NUM>X, where X is -OR' or -NR'R", R' and R" each independently being hydrogen or an alkyl, n is <NUM> or a positive integer, and the wavy line indicates covalent attachment to the peptide.

In another aspect, the present disclosure is generally directed to a method as defined in the claims. In one set of aspects, the method includes noncovalently binding a europium ion to a peptide comprising a first portion, and a second portion comprising a phosphate group, where the first portion has a structure:
<CHM>
where each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently is hydrogen or -SO<NUM>X such that at least one of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is -SO<NUM>X, where X is -OR' or -NR'R", R' and R" each independently being hydrogen or an alkyl, n is <NUM> or a positive integer, and the wavy line indicates covalent attachment to the peptide; and determining luminescence of the structure to determine binding of the europium ion to the first and second portions of the peptide.

The method, in accordance with another set of aspects, includes exposing a kinase to a peptide as defined in the claims and magnesium ions, where after phosphorylation of the peptide by the kinase, the magnesium ions non-covalently bind to the peptide to form a complex; exposing the complex to europium ions; and determining phosphorylation of the peptide by determining luminescence of the structure at <NUM> +/- <NUM>.

In still another set of aspects, the method includes exposing a solution suspected of containing a phosphatase to a phosphorylated peptide as defined in the claims and magnesium ions; exposing the complex to europium ions; and determining luminescence of the complex to determine dephosphorylation of the peptide by the phosphatase.

The method, in yet another set of aspects, includes exposing a phosphatase to a peptide comprising a first portion, and a second portion comprising a phosphate group, where the first portion has a structure:
<CHM>
where each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently is hydrogen or -SO<NUM>X such that at least one of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is -SO<NUM>X, where X is -OR' or -NR'R", R' and R" each independently being hydrogen or an alkyl, n is <NUM> or a positive integer, and the wavy line indicates covalent attachment to the peptide; and determining dephosphorylation of the peptide by determining luminescence of the structure at <NUM> +/- <NUM>.

In another aspect, the present disclosure encompasses methods of making one or more of the aspects described herein. In still another aspect, the present disclosure encompasses methods of using one or more of the aspects described herein.

The present disclosure generally relates to assays of enzymes responsible for phosphoregulation including kinases (such as protein kinases, which mediate protein and peptide phosphorylation) and phosphatases (such as protein phosphatases, which mediate protein and peptide dephosphorylation). Certain aspects of the disclosure use europium ions that exhibit chelation-enhanced luminescence. Phosphorylation of a peptide by a kinase may cause a complex to form between the europium ion, the phosphate group, and a reporter group such as a hydroxyquinoline, which results in luminescence when in the complexed state. Thus, in certain aspects, determination of luminescence may be indicative of kinase activity. Certain aspects also include the use of substrates for detection of phosphatase activity, where dephosphorylation results in a loss of signal assay. Other aspects are generally related to techniques for making and using such peptides or complexes, kits involving such peptides or complexes, and the like.

One aspect of the disclosure is now described with respect to <FIG>. In this figure, a peptide <NUM> is exposed to a kinase, such as protein kinase <NUM>, which transfers a phosphoryl group <NUM> to the peptide, e.g., on certain amino acid residues such as serine, threonine, or tyrosine. In certain aspects, however, determination of the phosphorylation event, qualitatively and/or quantitatively, may be desired. In addition, it should be understood that in other aspects, a phosphatase may be used.

In some cases, such as is shown in <FIG>, this may be accomplished by using a group such as a hydroxyquinoline and ions that can non-covalently coordinate with the reporter group and the phosphate. Thus, in this figure, a lanthanide (III) [Ln(III)] ion is complexed to both phosphate <NUM> and reporter group <NUM> on peptide <NUM>.

One non-limiting example of such a compound is illustrated in <FIG>, with europium (III) [Eu(III)] as the lanthanide ion. In this figure, peptide <NUM> includes both a phosphate group <NUM> and a hydroxy-quinoline group <NUM>. Eu(III) is non-covalently coordinated to both of these groups. Upon application of incident light <NUM> of a suitable wavelength, the complex is able to luminesce, producing emissive light <NUM> that can be determined in some fashion, e.g., which can be used to determine that the peptide has been phosphorylated or not (for example, by a kinase or a phosphatase). However, if no phosphate group <NUM> is present, then the complex is not able to form (or forms poorly), and emissive light <NUM> is not produced (or is produced, but at a much weaker signal). Accordingly, for instance, the presence of a kinase or a phosphatase able to phosphorylate the peptide can be determined, e.g., qualitatively and/or quantitatively.

Also, such systems are not limited to only the detection of kinases such as protein kinases. For instance, in one aspect, such a system could be used to determine whether a phosphate is present on a structure or not. In another aspect, a phosphopeptide could be used to measure the activity of a protein phosphatase. In yet another aspect, the system may be used to determine a phosphatase.

In some cases, the complex, when formed, can be determined by a luminescence signal. For example, if the complex is present, then incident light of a suitable wavelength may cause the complex to luminesce and produce emissive light that can be determined, e.g., qualitatively or quantitatively.

For example, the complex may have a structure:
<CHM>.

In this structure, the phosphate is at the bottom and the reporter group ( hydroxyquinoline group) is at the top, both chelated to a lanthanide ion (Ln). The wavy lines indicate attachment to a peptide.

In addition, it should be understood that Ln may also complex to other ligands, such as H<NUM>O (not illustrated here for purposes of clarity). For example, the Ln may form complexes that coordinate to <NUM>, <NUM>, <NUM>, <NUM>, or more ligands in all. Thus, for example, the Ln above may also be coordinated to <NUM>, <NUM>, <NUM>, <NUM>, or more H<NUM>O molecules, e.g., in addition to the bonds that are shown.

There may be any suitable number of amino acid residues between the two attachment points. For example, there may be <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> or more amino acid residues between the two attachment points, and either one may be closer to the N-terminus or the C-terminus. The peptide itself may have any suitable number of amino acid residues. For example, the peptide may have at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, at least <NUM>, or at least <NUM> amino acid residues. In some cases, the peptide may have less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, or less than <NUM> amino acid residues. The amino acids may include naturally-occurring and/or synthetic amino acids.

As mentioned, a second portion of the common structure includes a moiety able to be phosphorylated, e.g., to introduce the phosphate group shown above. For instance, the second portion may include serine, threonine, or tyrosine, wherein the phosphoryl group is transferred to the oxygen in the amino acid side chain. In some cases, the second portion may include a naturally-occurring or synthetic amino acid residue that includes a hydroxyl group in the side chain. In addition, in certain aspects, the second portion can include histidine, lysine, or arginine (which may be phosphorylated through phosphoramidate bonds), or aspartic acid or glutamic acid (which may be phosphorylated through mixed anhydride linkages). Thus, as a non-limiting example, a phosphorylatable amino acid such as serine, threonine, or tyrosine may have a free -OH group such as that shown in <FIG> and defined as Sox-OH in SEQ ID NO: <NUM>; upon phosphorylation (e.g., by a kinase), the OH may be converted to a -OPO<NUM>= phosphate group (i.e., as -OPO<NUM>= as shown in this figure and defined as Sox-P in SEQ ID NO: <NUM>).

The lanthanide ion is europium. In most cases, the Europium within the complex will be in the +<NUM> state, but may be in the +<NUM> state.

The first portion of the complex is a reporter group such as a hydroxyquinoline.

The quinoline may have a structure:
<CHM>
where the wavy line indicates covalent attachment to a peptide or other common structure, e.g., as discussed below. In some cases, chelation, coordination or non-covalent binding of a europium ion may occur with the oxygen and the nitrogen atoms. In addition each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently can be hydrogen or a -SO<NUM>X moiety such that one, two, or more, of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is a -SO<NUM>X moiety. X in such structures may be -OR' or -NR'R", where R' and R" each independently is hydrogen or an alkyl. R' and R" may be the same or different from each other. The alkyl group may have <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or more carbon atoms, may be unsubstituted or substituted, cyclic or linear, and may be saturated or unsaturated. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, cyclopentyl, (cyclohexyl)methyl, cyclopropylmethyl, etc. Non-limiting examples of substituted alkyls include haloalkyls, thioalkyls, aminoalkyls, chloromethyl, fluoromethyl, trifluoromethyl, <NUM>-chloroethyl, <NUM>-chloroethyl, etc..

As a non-limiting example, the hydroxyquinoline has a structure:
<CHM>
i.e., where R<NUM>, R<NUM>, R<NUM> and R<NUM> are each hydrogen, and R<NUM> is -SO<NUM>X, where X is NR'R" and each of R' and R" is independently methyl. In other aspects, the hydroxyquinoline may have structures such as:
<CHM>
In addition, as previously mentioned, two or more -SO<NUM>X groups may be present, e.g., as substituents on the quinoline or other reporter group in some cases.

As mentioned, the reporter group is attached to a common structure, such as a peptide, at the location indicated by the wavy line. In some aspects, there may also be one or more linkers within the reporter group, such as one or more -CH<NUM>- groups, to facilitate attachment, i.e., as in the structure:
<CHM>
where n is <NUM> or a positive integer (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, etc.).

In some aspects, the reporter group may be a side chain of an amino acid residue within the peptide or protein (i.e., R in the backbone structure. -NH-CHR-C(=O)-. (Thus, this may have a structure similar to glycine, in which alpha -CH is replaced by the reporter group. ) In another set of aspects, the reporter group may be attached to a side chain of an amino acid residue. For example, the reporter group may be covalently bonded to the sulfur of a cysteine residue within the peptide or protein, forming a disulfide linkage (e.g., upon reaction of a thiol within the reporter group to the thiol group within the cysteine) or a thioether linkage (e.g., upon reaction of a haloalkyl group within the reporter group to the thiol group within the cysteine), for instance, as is shown in the example of <FIG>. As another non-limiting example, reaction of an amine within the reporter group to a carboxylic acid can be used to form an amide linkage, e.g., to amino acid residues such as aspartic acid or glutamic acid.

Accordingly, as a non-limiting example, the reporter group may have a structure:
<CHM>
for example, where the -(CH<NUM>)n- group is bound to the sulfur of a cysteine residue, to a disulfide group (e.g., of a cysteine residue), to an amide linkage, to a backbone structure (e.g., as R in the backbone structure. -NH-CHR-C(=O) -. ), or the like. The reporter group may be part of a common peptide or other common structure. A non-limiting example of such a peptide is LVEPLTPCGEA (SEQ ID NO: <NUM>), discussed in more detail in Example <NUM>.

Accordingly, as mentioned, a complex may be formed if the reporter group, a phosphate derivative, and a europium ion are present. As a non-limiting example, a complex such as the following may be formed (again, coordination to other ligands, such as H<NUM>O, are not illustrated here for purposes of clarity):
<CHM>
Eu(III) may, for example, may be <NUM> or <NUM> coordinate. Such a complex can accordingly be used to determine, for example, if Eu(III) is present, and/or to determine if a phosphorylation has occurred. As a non-limiting example, as certain amino acids can be phosphorylated upon reaction with a kinase (e.g., a protein kinase), the presence of this complex may be used to determine the presence and/or activity of kinases.

The concentrations of the peptide (or other structure) may be, for example, between about <NUM> micromolar to <NUM>. For example, the concentration may be at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> micromolar, at least <NUM> millimolar, at least <NUM> millimolar, and/or no more than <NUM> millimolar, no more than <NUM> millimolar, no more than <NUM> millimolar, no more than <NUM> micromolar, no more than <NUM> micromolar, no more than <NUM> micromolar, no more than <NUM> micromolar, no more than <NUM> micromolar, no more than <NUM> micromolar, or no more than <NUM> micromolar, including combinations of any of these ranges. Any suitable phosphoryl donor source may be used, e.g., adenosine <NUM>'-triphosphate (ATP), for example, at concentrations between <NUM> micromolar and <NUM> millimolar. However, other concentrations can be used in other aspects. Those of ordinary skill in the art will be able to prepare such peptides, e.g., using techniques such as standard (N-alpha)-Fmoc-amino acid protection chemistry together with standard solid-phase peptide synthesis.

In some cases, protection and selective deprotection of amino acids may be used, e.g., to modify a side chain or a residue. For example, orthogonal side-chain protection techniques may be used, such as the O-allyl ester (OAll) (e.g., for the carboxyl group in the side chain of glutamic acid or aspartic acid, for example), the allyloxy carbonyl (Alloc) (e.g., for the amino nitrogen in the side chain of lysine or ornithine, for example), p-methoxytrityl (MMT) or acetamidomethyl (Acm) (e.g., for the sulfhydryl of cysteine). As non-limiting examples, OAll and Alloc can be removed by Pd, Acm can be removed by iodine treatment, and MMT can be removed by mild acid treatment.

Examples of kinases include, but are not limited to, serine/threonine and tyrosine kinases. The concentration of the kinase can range from, as non-limiting examples, about <NUM> picomolar to about <NUM> micromolar. Exemplary kinases include cAMP dependent protein kinase, protein kinase C, Ca/calmodulin dependent kinases, AMP activated kinase, s6 kinases, eIF-<NUM> kinases, p34cdc2 protein kinase, mitogen activated protein kinases, casein kinase-<NUM>, casein kinase-<NUM>, glycogen synthase kinase-<NUM>, Tyr-specific protein kinases. Other non-limiting examples of protein kinases include AGC kinases (e.g., PKA, PKC, PKG, etc.), CaM kinases (calcium/calmodulin-dependent protein kinases), CK1 kinases (casein kinase <NUM>), CMGC kinases (e.g., CDK, MAPK, GSK3, CLK kinases), STE kinases (yeast Sterile kinases), TK kinases (tyrosine kinases), TKL kinase (tyrosine-kinase like kinases). Protein kinases also include serine/threonine-specific protein kinases (e.g., CK2, PKA, PKC, Mos/Raf kinases, MAPKs, CaM kinases, phosphorylase kinase, PKB, IRAK-<NUM>, etc.), tyrosine-specific protein kinases (e.g., PDGFR, EGFR, IGF1R, SCF, etc.). Still other examples of kinases include, but are not limited to, lipid kinases, carbohydrate kinases, nucleoside-phosphate kinases, and nucleoside-diphosphate kinases. In addition, it should be understood that the disclosure is not limited to only kinase detection. For instance, in certain aspects, the state of phosphorylation of a peptide or other structure may be desired, i.e., not necessarily one that has been exposed to a kinase.

In addition, as previously discussed, certain aspects of the present disclosure are generally directed to phosphatases, including tyrosine phosphatases, serine phosphatases, or the like. In some cases, the phosphatases are relatively promiscuous, and may recognize more than one substrate.

As mentioned, the complex, if present, can be determined by luminescence emission in accordance with certain aspects. In some cases, the complex may be determined, qualitatively or quantitatively, by determining light emitted by the complex when exposed to incident light of a suitable wavelength. Any method known to those of ordinary skill in the art can be used to determine luminescence, including, for example, fluorimeters, spectrofluorimeters, fluorescence plate readers, photomultiplier tubes, avalanche photodiodes, or the like.

In some cases, incident light of around <NUM> may be used to stimulate the complex. The incident light may have a wavelength of, for example, <NUM> +/- <NUM>, <NUM> +/- <NUM>, or <NUM> +/- <NUM>. In some cases, light of other frequencies (or frequency distributions) may be used.

Europium ions are due to their emission frequencies, and/or their lifetimes (i.e., after excitation). For example, in certain cases, europium ions may exhibit emission wavelengths of around <NUM> to <NUM> and around <NUM> to <NUM>. For instance, the emission may be monitored around <NUM>, e.g., <NUM> +/- <NUM>, <NUM> +/- <NUM>, or <NUM> +/- <NUM> or at other relevant Eu(III) luminescence emission bands. Such wavelengths may allow for surprisingly little background interference, e.g., from other organic or biological species.

In addition, chelated europium ion complexes may exhibit lifetimes on the order of microseconds to milliseconds, in contrast to non-lanthanide ions and organic fluorophore that may exhibit lifetimes on the order of nanoseconds. The longer lifetimes may thereby allow for easier detection. For example, in some instances, the longer lifetimes can allow emissions from interfering background organic or biological species to be "gated" out, for example, by using a short time delay before determining the lanthanide ion luminescence. Accordingly, complexes comprising europium ions may be easier to detect in certain aspects than other, non-lanthanide ion chelates.

In some aspects, the europium ion may directly bind non-covalently to or chelate with the reporter group and the phosphate group, e.g., to form structures such as those shown herein. For example, a solution may contain a peptide (or other structure as discussed herein) suspected of being phosphorylated (e.g., upon exposure to a kinase). The peptide may also contain a reporter group. The solution may also contain europium ions, which can be added when the solution is formed, or afterwards. For example, such ions may be added before or after exposure of a peptide or other structure to a kinase, or a phosphatase. In some cases, binding of europium ions to form a complex may occur spontaneously, e.g., under ambient temperatures and/or pressures.

In some aspects, a complex may initially be formed using other ions, which ions may then be exchanged or replaced with europium ions. For instance, in some cases, an ion such as magnesium may first complex to the reporter group and the phosphate group, and the ion may then be exchanged or replaced with europium ions.

A non-limiting example of such a reaction is shown in <FIG>. The other ion and the europium ion may both be present initially, or in some cases, the europium ion may be added after formation of the complex. The ion exchange within the complex may be partial or total, i.e., <NUM>% or a smaller percentage of the ions within the complex may be replaced by europium, depending on factors such as ion concentration, temperature, duration, or the like.

The methods as discussed herein can be used in vitro or in vivo. In some applications, a reaction may be conducted in a buffer containing suitable ions, phosphates, and/or kinases, phosphatases, etc., depending on the application. Suitable buffers include, but are not limited to, HEPES, MES, TRIS, or the like.

In some cases, the reaction may occur inside a cell. The cell may include sufficient kinases, phosphatases, Mg<NUM>+, ATP sources, etc., e.g., in the cytosol. In some cases, additional ions, such as europium or other lanthanides may also be added. In certain aspects, cellular internalization sequences can be used. Non-limiting examples of cellular internalization sequences include penetratins, HIV-TAT domains and poly-arginine sequences. However, it should be understood that cells are not required, and in some aspects, reactions such as those discussed herein may occur in vitro.

In addition, in some aspects, the hydroxyquinoline-modified phosphopeptides as their europium ion chelates or other phosphopeptide-lanthanide ion complexes can be used as phosphatase substrates. In some cases, phosphatase activity will remove the phosphate and as a result, luminescence will decrease due to reduced affinity for the lanthanide; thus, some or all of the hydroxyquinoline-modified peptides that serve as kinase substrates can similarly serve as phosphatase substrates once phosphorylated in certain aspects.

In any of the amino acid sequences described herein, it should be understood that the sequence can have any suitable C-terminus, for example, one that is capped with an amide (e.g., a primary amide), or is not capped; and independently, the sequence can have any suitable N-terminus, for example, one that is free, or is N-acetylated.

This example illustrates selective protein kinase activity assays using a fluorescent readout at <NUM> due to the formation of an Mg(II)-bound CSox-phospho-peptide chelate. See also <FIG>.

The same peptides can also be applied in a quenched point readout, e.g., at the end of a kinetic analysis where the assay mixture is added to a buffer containing Eu(III) ions, regardless of whether the enzyme reaction has been stopped or not. The Eu(III) displaces the Mg(II) and forms a new chelate that shows sensitized emission with a maximum luminescence around <NUM>. This is at a wavelength where there may be little or no background interference. Due to the relatively long lifetime of the Eu(III) chelate, any emission from any interfering background fluorescent organic species may also be "gated" out in certain aspects by applying a short delay (e.g., <NUM> to <NUM> microseconds or longer) before collecting the Eu(III) luminescence data.

This example illustrates Sox-Eu peptide sensors to probe kinase activity, in accordance with certain aspects of the disclosure.

<FIG> shows schematic representations of a Eu(III)-based Sox sensor to probe protein kinase activity in this example. The Sox-containing substrate is silent, but upon phosphorylation, the chromophore can bind Eu(III) and undergo chelation-enhanced luminescence.

The instrumentation used in this example is as follows. The UV-Vis spectrophotometer was a NanoDrop ND-<NUM> (Labtech). The fluorimeter was a CLARIOstar® spectrofluorimeter equipped for time-resolved fluorescence measurements (BMG LABTECH). The plate was a Greiner assay plate (Greiner Bio-One), <NUM>-well, no lid, flat bottom, medium binding surface, non-sterile, black polystyrene.

For the stock solutions, only reagents of the highest purity and lowest metal ion content were used. Stock solutions of the peptides were prepared in doubly deionized water and concentrations were determined by UV-Vis (based on the determined extinction coefficient of the fluorophore unit, <NUM>-(N,N-dimethylsulfonamido)-<NUM>-hydroxy-<NUM>-methylquinoline, ε<NUM> (epsilon) = <NUM>-<NUM> cm-<NUM> at <NUM> in <NUM> NaOH with <NUM> Na<NUM>EDTA). An average of the values from three separate solutions, each prepared using a different volume of the stock solution, was read on the UV-Vis spectrophotometer. Purified peptide stock solutions could be stored at <NUM> for at least <NUM> months or -<NUM> for longer periods.

A europium chloride (EuCl<NUM>) stock solution of <NUM> (<NUM>% trace metals basis, Aldrich) was prepared in ultrapure water. <NUM> HEPES (SigmaUltra) was prepared and adjusted to pH <NUM> with NaOH (<NUM>+%, Aldrich) solution. <NUM> NaCl was prepared by dissolving sodium chloride (SigmaUltra) in ultrapure water.

The luminescence experiments were conducted as follows. Time-resolved emission measurements were made with a CLARIOstar® spectrofluorimeter. The spectra were recorded between <NUM> and <NUM>, and all measurements were made using the following settings: excitation wavelength <NUM>; delay time <NUM>; stepwidth <NUM>; emission bandwidth <NUM>; gain: <NUM>; focal height <NUM>; <NUM> flashes per well. All of the spectra were corrected for background luminescence by subtracting a blank scan of the solvent solution.

Comparison of phosphopeptide (Sox-P) and unphosphorylated biosensor (Sox-OH). Time-resolved luminescence spectra of the phosphopeptide Sox-P (<NUM> micromolar) and the corresponding unphosphorylated biosensor Sox-OH (<NUM> micromolar) at <NUM>, in <NUM> HEPES buffer, pH <NUM>, <NUM> NaCl, were recorded in the fluorometer. The difference in luminescence was determined by comparing the luminescence intensity at the maximum emission wavelength (<NUM>) of synthetic phosphopeptide and unphosphorylated peptide. The reported values are averages of three separate experiments.

<FIG> shows luminescence difference between the phosphorylated peptide (Sox-P) and the corresponding substrate (Sox-OH) using different concentrations of EuCl<NUM>. <FIG> shows a comparison of the spectra of phosphorylated (Sox-P, <NUM> micromolar) and the unphosphorylated peptide (Sox-OH, <NUM> micromolar) in the presence <NUM> micromolar of EuCl<NUM>.

Determination of Eu(III) dissociation constants (KD). Eu(III) titrations were performed at <NUM> in a buffer containing <NUM> HEPES buffer, pH <NUM>, <NUM> NaCl, and <NUM> micromolar of the of the phosphopeptide Sox-P (<NUM> micromolar) or the corresponding unphosphorylated biosensor Sox-OH (<NUM> micromolar) in a total volume of <NUM> microliters. Aliquots of EuCl<NUM> stock solutions were added (for the final EuCl<NUM> concentration in the well to be in the range of approximately <NUM>-<NUM> micromolar) and the data was recorded in the fluorometer. Data were fit with the program GraFit <NUM>. The reported values are averages of three separate experiments.

<FIG> shows Eu(III) titration curves for Sox-OH and Sox-P. Table <NUM> shows peptide Sequences and Dissociation Constants for Eu(III).

This example shows an illustration of binding using a CSox substrate for detecting the activity of certain tyrosine kinases. The instrumentation used in this example was a Biotek Synergy Neo2 microplate reader. In this reaction, depicted in <FIG>, a <NUM> microsecond delay allows binding of Eu(III) to be determined within the complex as shown. Time-resolved fluorescence at ><NUM> emission allows for the elimination of compound interference. In particular, in this example, a kinase activity was determined by fluorescence using CSox peptide Ac-EEPIYVC(Sox)FG (SEQ ID NO: <NUM>), in combination with europium ions.

The reaction conditions used included <NUM> HEPES (pH <NUM>), <NUM>-<NUM> micromolar ATP, <NUM> DTT, <NUM>% Brij-<NUM>, <NUM>% glycerol, <NUM>/ml BSA, <NUM>-<NUM> MgCl<NUM>, <NUM> micromolar CSox peptide, <NUM>-<NUM> YES (a kinase), amino acids <NUM>-<NUM> (Carna Bio).

The reaction was set up using <NUM> microliters CSox-peptide (5x), <NUM> microliters reaction mix with ATP & DTT (<NUM>. 67x), <NUM> minute preincubation (all components except EDB/YES) at <NUM>, <NUM> microliters enzyme dilution buffer (1x) or YES (5x in EDB) to <NUM>% glycerol final in a <NUM> microliter final reaction volume.

Reads were performed kinetically with an excitation wavelength of <NUM> and an emission wavelength of <NUM> (filter, <NUM> or <NUM> bandwidth), and measuring fluorescence intensity from the top of the plate. For the endpoint assay, following addition of europium ion (<NUM> to <NUM>,<NUM> micromolar), the fluorescence signal was determined using an excitation wavelength of <NUM> and an emission wavelength of <NUM> (filter, gain = <NUM>) or as wavelength scan from <NUM> to <NUM>, using time resolved-fluorescence (<NUM> msec delay) and a gain of <NUM>.

The enzyme dilution buffer (EDB) was <NUM> HEPES, pH <NUM>, <NUM>% Brij-<NUM>, <NUM>% glycerol, <NUM> DTT, and <NUM>/mL Bovine Serum Albumin (BSA). Thus, the actual final reaction concentrations were <NUM> HEPES, pH <NUM>, <NUM>% Brij-<NUM>, <NUM>% glycerol, <NUM> DTT, and <NUM>/mL BSA. All components except the enzyme were equilibrated to <NUM>. The reactions were run in Corning, half-area <NUM>-well, white flat round bottom polystyrene NBS microplates after sealing using optically-clear adhesive film (TopSealA-Plus plate seal, PerkinElmer, applied with a roller) to eliminate evaporation and resulting drift.

<FIG> show results for Eu(III) titration using <NUM>-<NUM> micromolar using <NUM> YES & CSox substrate Ac-EEPIYVC(Sox)FG (SEQ ID NO: <NUM>). The reaction was run for <NUM> minutes with <NUM> YES, <NUM> micromolar CSox substrate, <NUM> MgClz, <NUM> micromolar ATP, and <NUM>-<NUM> micromolar europium. Signals in the presence of Eu(III) were much greater than blanks or controls, with generally greater signals at higher concentrations. Z' values were greater than <NUM>, considered to be an excellent assay, throughout the range tested.

This example shows the application of a hydroxyquinoline-sensitized Eu chelate with an acetyl and primary amide-capped tridecapeptide with a Thr-Val-CSox-Ala-Leu (SEQ ID NO: <NUM>) core to determine the IC<NUM> of staurosporine using a YES kinase. A comparison kinetic vs endpoint (Eu) assay is shown in <FIG>. The reaction was performed for <NUM> minutes with <NUM> YES, <NUM> micromolar AQT0104, <NUM> MgClz, <NUM> micromolaor ATP, and then <NUM> micromolar of Eu was added. These results were consistent with the values reported for YES using a PE/caliper MBS Assay.

This example illustrates the application of a hydroxyquinoline-sensitized Eu chelate with an acetyl and primary amide-capped tridecapeptide with a Tyr-Arg-CSox-Pro-Ser (SEQ ID NO: <NUM>) core to determine the IC<NUM> of staurosporine IC50 using a CaMK2δ kinase. A comparison kinetic vs endpoint (Eu) assay is shown in <FIG>. In both figures, the concentrations were as follows: CaMK2δ kinase, <NUM>; ATP, <NUM> micromolar; and peptide, <NUM> micromolar.

This example illustrates the application of a hydroxyquinoline-sensitized Eu chelate with an acetyl and primary amide-capped hexadecapeptide with an Ac-CSox-Gly-Thr-Phe (SEQ ID NO: <NUM>) core to assess activity of ASK1 kinase at low and high ATP concentrations. The results are shown in Fig. 10A and 10B. High S/B (signal to background) (<NUM> or higher) and Z' (<NUM> for low ATP and <NUM> for high ATP conditions) were observed.

Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc..

Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc..

When the word "about" is used herein in reference to a number, it should be understood that still another aspect of the disclosure includes that number not modified by the presence of the word "about.

Claim 1:
A composition, comprising:
a peptide having a first portion comprising a structure:
<CHM>
wherein each of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> independently is hydrogen or -SO<NUM>X such that at least one of R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> is -SO<NUM>X, wherein X is -OR' or -NR'R", R' and R" each independently being hydrogen or an alkyl, and n is <NUM> or a positive integer, and
a second portion comprising a phosphate group,
wherein the N and/or the O of the first portion, and the phosphate group of the second portion, are coordinated via a europium ion.