CA2553218A1 - Fluorescence-based adp detection system - Google Patents

Abstract

A method and kit for detecting a nucleotide, or differentially detecting one nucleotide in a mixture of at least two nucleotides, in a solution comprising the steps of adding a chelator to the solution and detecting a signal created or altered upon the addition of the chelator using a spectroscopic detection system. A lanthanide may also be added to the solution before the detection of a signal. Preferably, the spectroscopic detection system is a fluorescence-based system.

Description

FLUORESCENCE-BASED ADP DETECTION SYSTEM
FIELD OF THE INVENTION
[0001JThe present invention is in the field of nucleotide detection. In particular, it relates to the differential spectroscopic detection of nucleotides.
BACKGROUND OF THE INVENTION

(0002] In the last few years protein kinases have become a target for drug development for pharmaceutical companies. It has been estimated that up to 20%
of all the targets in the pharmaceutical industry are currently kinases. The reason for this interest has to do with the fact that kinases play a crucial role in fundamental cellular processes. Disturbances in kinase activity may cause or be an indication of disease in humans, and targeting kinase molecules or receptors with drugs may alter the course of the disease.

[0003] Kinases are enzymes (biochemical catalysts) that transfer a phosphate group from ATP (adenosine triphosphate) to a substrate. As a result of the phosphate transfer, the ATP becomes dephosphorylated to form ADP (adenosine diphosphate).
Once the substrate is phosphorylated in vivo, a biochemical pathway may be activated.
A single kinase may have multiple substrates and, depending on which substrate is being phosphorylated, different pathways may be activated. Substrate selectivity is thus an important characteristic of kinases. The biochemical reaction that kinases carry out is the following:

[0004]ATP + Kinase + Substrate + ADP + Kinase + Substrate-P (I) [0005]One of the requirements of the reaction is that the substrate have an available hydroxyl group to accept the phosphate group being transferred by the kinase enzyme from the molecule of ATP. Polypeptide or protein substrates thus generally contain tyrosine (tyrosine kinases) or serine/threonine (serine/threonine kinases) as the acceptor amino acid.

[0006] Detection of kinase activity in the clinical setting plays an important role in evaluating various states of human health. Creatine kinase (GK), for example, is a "leakage" enzyme present in high concentration in the cytoplasm of myocytes and is the most widely used enzyme for evaluation of neuromuscular disease. Current methods for CK detection involve multiple steps including the use of various other enzymes.

[0007] Detection of kinase activity for the purposes of drug discovery and drug profiling presents an interesting challenge for the pharmaceutical industry. Coupled detection systems with multiple enzymes, which are currently used in industry, are not practical due to the need for counter screens to rule out the effects of drugs on enzymes other than the one of interest. For example, in looking for CK inhibitors, hexokinase (HK), a coupled enzyme, can be affected by drugs that interact with the ATP binding site of CK
because HK contains its own ATP binding site.

[0008] One approach that investigators searching for inhibitors of kinases in their drug discovery programs have traditionally used is the detection of substrate phosphorylation as a way to monitor kinase activity. The challenge is that the substrate for every kinase can be different and a single kinase may have multiple phosphorylation sites even within the same substrate. In every instance assay development has to be done in order to find the optimal conditions for the assay, which can be very time consuming. A currently used method to detect substrate phosphorylation is an ELISA
based assay in which the detection of the phosphorylated substrate is done using a specific antibody. ELISA based systems show great sensitivity but many steps are required, a typical assay may run for hours, and many manipulations are needed, increasing the chance for error.

[0009]Another method currently used to monitor kinase activity is the detection and quantitation of the decrease of ATP as the assay progresses. This method is limited by the production of the product - in this case ADP - which, when accumulated, may inhibit the activity of the kinase. Application of such a methodology requires extensive assay development.

[0010] Detection systems traditionally used to monitor the progress of kinase assays utilize spectrometric techniques such as fluorescence spectrometry, fluorescence polarization (Panvera, Molecular Devices, Chromagen), time-resolved fluorescence (Cis-Bio), absorbance spectrometry (MDS Pharma Services, Upstate), luminescence spectrometry (Promega), and non-spectrometric techniques such as scintillation (Perkin Elmer, Amersham) and chromatography (Caliper).
BRIEF DESCRIPTION OF THE DRAWINGS
[0011 ] Figure 1 shows superimposed fluorescence spectra of ADP and ATP with HEPES used as a control.
[0012] Figure 2 shows superimposed fluorescence spectra of ADP in the presence of EDTA (ADP-EDTA) and ATP in the presence of EDTA (ATP-EDTA) with HEPES-EDTA
used as a control.
[0013] Figure 3 shows superimposed fluorescence spectra of ADP in the presence of EGTA (ADP-EGTA) and ATP in the presence of EGTA (ATP-EGTA) with HEPES-EGTA used as a control.

[0014] Figure 4 shows the results of fluorescence data read at 500 nm showing a 50 mM EDTA solution upon titration with ADP.
[0015] Figure 5 shows the results of fluorescence data showing ADP in the presence of EDTA upon titration with Ca2+ and Mg2+.
[0016] Figure 6 shows the results of fluorescence data showing ADP in the presence of EDTA upon titration with ATP.
[0017] Figure 7 shows the results of fluorescence data showing a series of ADP
and ATP solutions of different concentrations upon addition of 50 mM of EDTA.
[0018] Figure 8 shows superimposed fluorescence spectra of selected nucleotides in the presence and absence of EDTA, with HEPES or HEPES-EDTA used as a control.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The inventors have developed technology for the detection of nucleotides in assays. It may be used as a means of monitoring biochemical activity in assays, such as, for example, kinase activity in a kinase assay. It is a spectroscopic detection system which provides a mechanism to study any activity in which the concentration of a diphosphorylated nucleoside changes with time. It can be used, for example, to monitor the conversion of a di- or triphosphorylated nucleoside to a mono- or diphosphorylated nucleoside respectively, or vice versa, independent of the substrate being used. A particular diphosphorylated nucleoside is ADP, and a particular triphosphorylated nucleoside is ATP. In addition to replacing currently used detection methods, this technology may be used to screen kinase targets against substrates for which there currently are no available detection methods. In the clinic or other situation, this methodology could be used to detect and/or monitor the activity of enzymes that utilize ATP and generate ADP.
[0020]ATP and ADP are invariably present in any kinase reaction. Systems based on monitoring the consumption of ATP and/or production of ADP can thus permit the monitoring of any kinase reaction, independent of the substrate. Since ADP is not present in the reaction before it starts, the monitoring of the production of ADP can be an effective method for monitoring the activity of a kinase.
[0021]The inventors have determined that there is a difference in the spectroscopic spectra of ATP and ADP which can be enhanced by the addition of a chelator.
More specifically, under certain circumstances, there is a fairly consistent difference in the fluorescence emission at around 450 to 550 nm in the fluorescence spectra of ADP and ATP molecules, as demonstrated in Figure 1. The inventors have additionally determined that, in the presence of a chelator such as ethylenediaminetetraacetic acid (EDTA) there is a substantial increase in ADP fluorescence in the 450 to 550 nm range without a corresponding increase in ATP fluorescence, as shown in Figure 2.
Similarly, as shown in Figure 3, there is a substantial increase in ADP fluorescence in the presence of the chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N,-tetraacetic acid (EGTA) without an increase in ATP fluorescence.
[0022] Figure 4 shows that the intensity of fluorescence at 500 nm is proportional to the amount of ADP added to a solution of EDTA.
[0023]As used herein, the term "chelator" refers to any molecule which possesses at least one functional group which can coordinate to a metal, either covalently or non-covalently. The chelator may be multidentate or coordinate in a unidentate manner.
The chelator may be a macrocycle such as a porphyrin. The chelator may chelate a metal by donating or sharing pi electrons. Chelators further derivatized to increase spectroscopic detection are also included. The functional group of the chelator may be negatively or positively charged, or neutral. Examples of suitable functional groups include carboxylato, thiolato, hydrido, cyano, carbonato, thiocarcamato, thiocarboxylato, thiophosphinato amino, phophoro, hydrazino, nitrilo, hydrazido, oxime, and thioether.
[0024] Examples of chelators include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene, and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).
[0025] Experiments showing that competitive displacement of EDTA from the ADP-EDTA interaction destroys the increased fluorescence in the ranges of 450 to 500 nm have also been conducted. As shown in Figure 5, there is a decrease in fluorescence at 500 nm when Mg2+ or Ca2+ is added to the reaction mixture, suggesting that the metal disrupts the interaction of ADP and EDTA. As well, as shown in Figure 6, an excess of ATP added to the reaction mixture attenuates the ADP-EDTA
interaction.
This result suggests that ATP may also directly interact with EDTA, but that the interaction does not cause an increase in fluorescence under these conditions.
[0026] Suitable spectrometric techniques include fluorescence spectrometry, ultraviolet and infrared absorption and transmission spectrometry, luminescence spectrometry, Raman spectrometry, and phosphorescence spectrometry. Most preferred is fluorescence spectrometry. Suitable fluorescence spectrometry techniques include the excitation of a sample with, for example, a xenon lamp, laser-induced fluorescence using lifetime fluorescence in order to increase detection sensitivity, fluorescent polarization which may boost the desired signal and lower background noise to improve sensitivity, and time-resolved fluorescence.

(0027] Optionally, metals, such as lanthanides, may be used to change the spectrometric characteristics, particularly the fluorescence spectrometric characteristics, of nucleotide-chelator interactions. It is known that tertiary complexes may be formed between certain organic molecules, chelators, and lanthanides (Diamandis EP, Christopoulos TK. Anal. Chem., 1990, 14:1149, Anal.
Christopoulos TK, Diamandis EP. Chem. 1992, 64: 342, hereby incorporated by reference). Upon formation of these tertiary complexes, an enhanced fluorescence signal was observed.
Before now, such complexes had not been observed between nucleosides or nucleotides and chelators and lanthanides where spectroscopic signals are enhanced.
[0028] The following experimental examples are illustrative of the use of this invention.
[0029] Experiments [0030] Experimental Details [0031 ] Spectrofluorometers [0032]The peak generated by a fluorescence signal is typically broad, and the ability to precisely select of a wavelength of excitation and/or emission largely depends on the calibration of the instrument and the fluorescence detection system, or reader, used.
For the experiments described herein, a Spex FluoroMax Spectrofluorometer (serial number 2093, Spex Industries, New Jersey) with a single well reader was used with a passband of 0.1 nm. In some cases, a Molecular Devices FLEXstation plate reader (serial number FX 01090, California) was used, which has a passband of 10 nm and therefore cannot detect the fluorescence signal as precisely as the SPEX
Fluoromax scanner.
(0033] Reagents [0034]The following reagents were used in the experiments described herein:
[0035]ADP (A-2754 Sigma Ultra, LOT#073k7007, Adenosine 5'-diphosphate sodium salt);
[0036]ATP ( A-7699 Sigma Ultra, LOT#053k7042, Adenosine 5'-triphosphate disodium salt);
(0037] EGTA (E0396 Sigma Ultra, Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid);
(0038] EDTA Sigma-Aldrich (E2-628-2 Ethylenediaminetetraacetic acid);
[0039]HEPES (J848, AMRESCO, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid);
[0040] CaCl2 (C-4901, Sigma, Calcium Chloride dehydrated); and [0041] MgCl2 (M8266, Sigma, Magnesium Chloride Anhydrous).

[0042] Abbreviations [0043] rfu = relative fluorescence unit (0044] Experiment 1: Difference in Fluorescence Spectra of ATP and ADP in the range of 450 to 500 nm [0045]A solution containing either 5 mM ADP or ATP in 10 mM HEPES pH 8.0 buffer was analyzed for fluorescence emission within the ranges of 400 to 600 nm using a SPEX Fluoromax fluorescence scanner. The excitation wavelength used for this experiment was 405 nm. This experiment shows that there is a weak fluorescence emission difference between ADP and ATP measured at around 500 nm.
[0046] Experiment 2: Selective Interaction of EDTA and EGTA with ADP over ATP
[0047]A fluorescence scan was performed using a SPEX Fluoromax on a solution containing 10 mM ADP or 10 mM ATP in 10 mM HEPES pH 8.0 buffer in the presence or absence of 50 mM EDTA. As shown in Figure 2, the fluorescence spectra of the solution containing ADP is greatly enhanced in the presence of EDTA. By contrast, ATP does not show an increase in fluorescence in the presence of EDTA.
Similarly, as shown in Figure 3, there is an increase in ADP fluorescence in the presence of EGTA
which is not seen in the ATP fluorescence. The peak seen at 475 nm is part of the background spectrum.
[0048] Experiment 3: ADP Titration in the Presence of EDTA
[0049]A solution 50 mM EDTA was titrated with ADP in 10 mM HEPES pH 8.0 buffer.
The fluorescence was read at 500 nm on a Molecular Devices FIexStation plate reader.
As shown in Figure 4, the data points resulting from the titration of the EDTA
solution with ADP follows a straight line over two log units. ADP below a concentration of 100 uM was beyond the limit of detection of the technique used.
[0050] Experiment 4: Addition of Ca2+ and Mg2+ to Solutions Containing ADP and EDTA
[0051] In this experiment, the effect of the addition of Ca2+ and Mg2+ to a solution containing 1 mM ADP and 30 mM EDTA in 10 mM HEPES pH 8.0 buffer was examined. As shown in Figure 5, the fluorescence emission signal generated by ADP
in the presence of EDTA was found to be reversed by the addition of 30 mM of Mg2+ or Ca2+, indicating that Mg2+ and Ca2+ can compete with the interaction of ADP
with EDTA. Fluorescence was monitored at 500 nm.
[0052] Experiment 5: Titration of Solution Containing ADP and EDTA with ATP
[0053] Since ATP does not seem to fluoresce in the presence of EDTA, and ADP
does, the effect of the presence of ATP on the EDTA-ADP induced fluorescence was examined. To a solution containing 5 mM ADP and 10 mM EDTA in 200 u1 of 10 mM

HEPES pH 8.0 buffer, increasing concentrations of ATP were added. The fluorescence was read at 500 nm using an excitation wavelength of 410 nm . The samples were read on a FIexStation (Molecular devices) plate reader. As shown in Figure 6, 10 mM
ATP
completely disrupts the interaction between ADP and EDTA. This observation suggests that ATP can directly interact with EDTA; however, it does not cause an increase in fluorescence under these conditions.
[0054] Experiment 6: Solution of ADP Titrated with EDTA
[0055] In this experiment, a series of solutions of ADP and ATP in 10 mM HEPES
pH
8.0 buffer at various concentrations were treated with 50 mM EDTA. HEPES-EDTA
was used as a control. Figure 7 shows the data where the fluorescence reading of the control subtracted from each data point.
[0056]These experiments establish that the sensitivity of detection of the ADP-chelator is about 1 uM by fluorescence spectroscopy.
[0057] Experiment 7: Effect of EDTA on Fluorescence of Nucleotides [0058]The fluorescence spectra of 5 mM solutions of ATP, ADP, guanidine diphosphate (GDP), and guanidine triphosphate (GTP) were taken in the absence and presence of 50 mM of EDTA. The fluorescence emission signals were analyzed between 450-600 nm. As shown in Figure 8, only ADP in the presence of EDTA
displays enhanced fluorescence, with a peak of fluorescence between 490-500 nm.
[0059] Experiment 8: Use of Lanthanides to Alter the Fluorescence Signal Generated by a Nucleotide in the Presence of a Chelator [0060] In order to establish the use of a tertiary complex formed between a lanthanide metal, a chelator, and a nucleotide to alter the fluorescence signal generated by the nucleotide in the presence of the chelator, the following experiments will be carried out.
[0061] (i) EDTA will be incubated in the presence and absence of terbium and in the presence or absence of increasing concentrations of nucleotides (ATP, ADP).
Fluorescence emission will be monitored with the use of a SPEX Fluoromax fluorescence scanner.
[0062] (ii) EDTA will be incubated with europium and in the presence or absence of increasing concentrations of nucleotides (ATP, ADP). Fluorescence emission will be monitored with the use of a SPEX Fluoromax fluorescence scanner.
[0063] (iii) A complex will be formed between an organic molecule such as salicylic acid, EDTA, and europium, yielding an increase in fluorescence. The effect of the addition of increasing concentration of nucleotides will be monitored by the use of SPEX Fluoromax fluorescence scanner.

[0064] Given the results obtained in 8(i), 8(ii), or 8(iii), conditions for monitoring changes in the ADP concentrations with time will be optimized.
[0065] While preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined by the following claims.
[0066]Although various examples of combined elements of the invention have been described, it will also be understood that these are not intended to be exhaustive and features of one embodiment may be combined with those of another, and such other combinations are contemplated to be within the scope of the invention disclosed herein.
[0067]All publications and other documents mentioned herein are hereby incorporated by reference into this document as though the entire contents thereof were reproduced herein.

Claims

[0068] We claim:
[0069] 1. A method of detecting a nucleotide in a solution, comprising the steps of (a) adding a chelator to the solution, and (b) detecting a signal created or altered upon the addition of the chelator using a spectroscopic detection system.
[0070] 2. The method of claim 1, wherein the nucleotide is adenosine diphosphate.
[0071] 3. The method of claims 1 or 2, wherein the spectroscopic detection system is a fluorescence-based system.
[0072] 4. The method of any of claims 1, 2, or 3, wherein the chelator is a polycarboxylate molecule.
[0073] 5. The method of claim 4, wherein the chelator is EDTA, EGTA, or a combination thereof.
[0074] 6. A method of differentially detecting one nucleotide in a mixture of at least two nucleotides in a solution, comprising the steps of (a) adding a chelator to the solution, and (b) detecting a signal created or altered upon the addition of the chelator using a spectroscopic detection system.
[0075] 7. The method of claim 6, wherein the nucleotide is adenosine diphosphate.
[0076] 8. The method of claims 6 or 7, wherein adenosine triphosphate is in the solution.
[0077] 9. The method of any of claims 6, 7, or 8, wherein the chelator is a polycarboxylate molecule.
[0078] 10. The method of claim 9, wherein the chelator is EDTA, EGTA, or a combination thereof.
[0079] 11. The method of any of claims 6 to 10, wherein the signal is a result of the interaction of adenosine diphosphate and EDTA, or EGTA or a combination thereof.
[0080] 12. The method of any of claims 6 to 11, wherein the spectroscopic detection system is a fluorescence-based system.
[0081] 13. The method of any of claims 6 to 12, wherein the signal is a fluorescence-based signal.
[0082] 14. The method of claim 13, wherein the fluorescence-based signal occurs between 450 and 550 nm.
[0083] 15. The method of any of claims 7 to 14, wherein the signal is increased in intensity compared to the fluorescence-based signal of adenosine diphosphate before the chelator(s) was added to the solution.

[0084] 16. The method of any of claims 1 or 15, wherein the signal is increased in intensity after the addition of the chelator.
[0085] 17. The method of any of claims 1 or 15, wherein the peak intensity of the signal is shifted after the addition of the chelator.
[0086] 18. The method of any of claims 1 or 15, wherein the signal is decreased in intensity after the addition of the chelator.
[0087] 19. A kit comprising (a) a suitable amount of chelator, and (b) instructions for conducting any of the methods according to any of claims 1 to 18.
[0088] 20. The method of any of claims 1 to 18 further comprising the addition of a lanthanide to the solution prior to step (b).
[0089] 21. The method of claim 20, wherein the lanthanide is terbium.
[0090] 22. The method of claim 20, wherein the lanthanide is europium.
[0091] 23. A kit comprising (a) a suitable amount of chelator, (b) a suitable amount of at least one lanthanide, and (c) instructions for conducting the methods according to any of claims 20 to 22.