WO2011056611A1 - Coupled reactions for analysis of nucleotides and their hydrolysis - Google Patents

Coupled reactions for analysis of nucleotides and their hydrolysis Download PDF

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WO2011056611A1
WO2011056611A1 PCT/US2010/054171 US2010054171W WO2011056611A1 WO 2011056611 A1 WO2011056611 A1 WO 2011056611A1 US 2010054171 W US2010054171 W US 2010054171W WO 2011056611 A1 WO2011056611 A1 WO 2011056611A1
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atp
sample
generated
amount
nmpk
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French (fr)
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Kirill Ostanin
Zhong-hua GAO
Scott G. Morham
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Myrexis, Inc.
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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
    • 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
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention generally relates to biochemical assays, and particularly to methods for the quantitative and qualitative analysis of nucleotides and their hydrolysis.
  • Nucleotides play many roles in living cells. In addition to their somewhat mundane role as the building blocks of the nucleic acids, nucleotides play vital and central roles in cellular metabolism and bioenergetics. Nucleotides (particularly nucleoside triphosphates, or NTPs) serve as the "energy currency" of the cell. In their high-energy phosphorylated forms, nucleoside triphosphates provide a source chemical energy for numerous metabolic transactions and transformations. Nucleotides (and nucleosides) also serve as structural and functional components of a variety of enzyme cofactors, as well as metabolic intermediates in a wide variety of enzymatic reactions. Finally, nucleotides (and nucleosides) serve as small-molecule messengers that can activate or facilitate the response of cells to hormones and a variety of extracellular stimuli.
  • biochemists As a consequence of the many important roles played by nucleotides in living systems, biochemists often desire to determine the types and amounts of nucleotides present both in vivo and in vitro, and measure their rates and extent of hydrolysis. In order to study various enzymatic reactions involving nucleotides, biochemists require simple and effective assays that allow for quantification of specific nucleotides, particularly just before, or immediately after, an enzymatic reaction occurs. Additionally, biochemists require assays that are specific for particular nucleotides that are involved in cellular signaling.
  • nucleotide hydrolysis assays are designed to measure adenosine triphosphate (i. e. , ATP)
  • this type of assay requires the use of sulfuric acid for color generation.
  • Sulfuric acid itself can cause spontaneous acid hydrolysis of nucleotides, thereby interfering with the assays by increasing background. Further, such acidic assay conditions are unsuitable for high-throughput screening (HTS) machineries.
  • HTS high-throughput screening
  • nucleoside monophosphates including nucleoside monophosphates, nucleoside diphosphates, nucleoside
  • the present invention provides a new approach and methods for
  • ribonucleotides and deoxyribonucleotides including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP.
  • the invention provides methods for the quantitative and qualitative analysis of a wide variety of nucleotides, other than ATP, and provides methods for quantifying their hydrolysis.
  • the methods and assays of the present invention are applicable to a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research. As a result of their ready adaptability and broad utility, these assays are of value to biochemists, molecular biologists and clinicians, among others.
  • the nexus of these methods and assays involves the coupling of an ATP- generating cyclic reaction referred to herein as a "Nucleotide Exchange Reaction” (or “NEPv”) to any suitable reaction that serves as a means of detecting the ATP generated by the NER (i. e. , the "ATP detection reaction” or “ADR”).
  • the NER comprises a reversible reaction catalyzed by a single enzyme. That enzyme is a Nucleoside
  • Monophosphate Kinase which, in one direction (the "forward” reaction), acts to transfer a high-energy phosphate from a substrate ribonucleoside triphosphate (NTP) other than ATP, or in some cases, a deoxyribonucleoside triphosphate (dNTP), to a ribonucleoside monophosphate (NMP) or dexoyribonucleoside monophosphate (dNMP), to form two corresponding nucleoside diphosphates (NDP or dNDP).
  • NTP substrate ribonucleoside triphosphate
  • dNTP deoxyribonucleoside triphosphate
  • NMP ribonucleoside monophosphate
  • dNMP dexoyribonucleoside monophosphate
  • the NMPK catalyzes the transfer of a phosphate group from a ribonucleoside diphosphate (NDP), or in some cases a deoxyribonucleoside diphosphate (dNDP), to ADP to form ATP and a corresponding NMP or dNMP.
  • NDP ribonucleoside diphosphate
  • dNDP deoxyribonucleoside diphosphate
  • NMPKs are a family of enzymes, the members of which have evolved to transfer phosphates between particular ribonucleotide triphosphates (NTPs) or, in some cases, a deoxyribonucleotidetriphosphates (dNTPs), and ADP.
  • NTPs ribonucleotide triphosphates
  • dNTPs deoxyribonucleotidetriphosphates
  • the NER can be configured to generate ATP in an NTP- (or dNTP-), NDP- (or dNDP-) or NMP- (or dNMP-) dependent fashion, where the NTP, NDP or NMP (or dNTP, dNDP or dNMP) are members of a particular cognate family of nucleotides that can serve as substrates for the NMPK being used.
  • NTP, NDP or NMP or dNTP, dNDP or dNMP
  • the NER can be configured such that the generation of ATP is dependent upon amount of GTP, GDP or GMP present.
  • the assay methods provided may be configured to qualitatively or quantitatively assay for the presence of specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, with the exception of ATP.
  • ATP cannot be specifically detected or quantified by the methods provided is that ATP is the particular nucleoside triphosphate generated by the NER, which is then detected and quantified by the ADR.
  • ATP generated by an NER can be detected by any suitable ATP detection method, so long as the ATP is either converted to ADP + inorganic phosphate (Pi) or AMP and pyrophosphate (PPi), or is otherwise removed from, or more generally consumed by the detection reaction (ADR).
  • ADP + inorganic phosphate (Pi) or AMP and pyrophosphate (PPi) or is otherwise removed from, or more generally consumed by the detection reaction (ADR).
  • ADR ATP detection method
  • luciferase enzyme that originally isolated from the firefly, Photinus pyralis.
  • This second reaction is specific for ATP because firefly luciferases specifically utilize ATP, and no other nucleoside triphosphates, as a substrate in the reaction that generates light.
  • U.S. patents 5,583,024; 5,674,713 and 5,700,673 provide methods for the recombinant expression of luciferase derived from bioluminescent insects, specifically bioluminescent beetles of the order Coleoptera.
  • U.S. patent 6,503,723 provides methods for detecting ATP in a sample using reactions catalyzed by luciferases that generate light, wherein the amount of light generated is indicative of the presence or absence, and the quantity, of ATP in the sample. The teachings of these four U.S.
  • the present invention can also be used to quantify the amount of nucleotide hydrolysis (other than ATP
  • nucleoside diphosphate or monophosphate that can be used as a substrate by a particular NMPK.
  • a particular enzyme such as an NTPase
  • the method works as follows: Nucleotide hydrolysis catalyzed by an enzyme under study (i. e. , an NTPase) generates a substrate for a particular matched NMPK (i. e. , an NMPK that can use that substrate) that, in turn, catalyzes an NER that generates ATP from ADP in a substrate-dependent fashion.
  • ATP so-generated is then utilized by luciferase to produce a bioluminescent signal and AMP + pyrophosphate (PPi).
  • PPi AMP + pyrophosphate
  • the disclosed NER-based methods have been successfully applied to determine the presence and relative amounts of GTP, GDP, CMP, ADP, AMP, UDP and UMP, but can be used for the qualitative and quantitative analysis of many other types of NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs.
  • the disclosed NER-based methods have also been used to develop an assay to measure the GTPase activity of a particular GTPase, and this assay has been successfully employed in a high-throughput screening (HTS) format.
  • HTS high-throughput screening
  • the disclosed NER-based methods can readily be applied to the study of other GTPases, and any other enzyme that catalyze reactions which generate specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, other than ATP.
  • the disclosed NER-based methods can also be used for the qualitative and quantitative analysis of specific cyclic nucleotides (i. e. , cAMP, etc.). Assays based upon the methods of the present invention have been found to be convenient, sensitive, robust and economical.
  • the disclosed NER-based assays may be used for the detection and quatification of a wide range of biologically- relevant nucleotides (other than ATP), and their hydrolysis.
  • the disclosed NER-based assays may be advantageously employed in a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research.
  • Figure 1 schematically depicts a cyclic Nucleotide Exchange Reaction (NER) catalyzed by an NMPK, wherein a phosphate group is ultimately transferred from "BTP" (top left; wherein BTP refers to any suitable ribonucleoside triphosphate other than ATP, or any suitable deoxyribonucleoside triphosphate, including dATP) to ADP, to generate ATP (bottom left);
  • NER Nucleotide Exchange Reaction
  • Figure 2 shows how an NER may be coupled with an ATP detection reaction (ADR) to create assays for the detection and quantification of a wide variety of nucleoside mono-, di-, and triphosphates;
  • ADR ATP detection reaction
  • Figure 3 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NMPs or dNMPs;
  • Figure 4 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NDPs or dNDPs;
  • Figure 5 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NTPs, other than ATP, and dNTPs (i.e. , "BTPs");
  • Figure 6 shows how the coupled reactions of Figure 2 may be used for the detection and quantification of cyclic nucleotides, when further coupled with a decyclization reaction catalyzed by a cyclic nucleotide phosphodiesterase;
  • Figure 7 depicts one configuration of the coupled reactions of Figure 2 being used for the quantification of the activity of a GTPase, particularly when the GTPase has a substantially greater affinity for GTP than the GMPK being used to catalyze the NER;
  • Figure 8 depicts another configuration of the coupled reactions of Figure 2 being used for the quantification of the activity of a GTPase, particularly by employing an excess of dATP as a substrate for the GMPK being used to catalyze the NER (Note: dATP is not a substrate for firefly luciferase);
  • Figure 9A depicts the amplification of the coding region of E. coli GMPK in preparation for cloning
  • Figure 9B depicts the induced expression
  • Figure 10A depicts the amplification of the coding region of E. coli CMPK in preparation for cloning
  • Figure 10B depicts the induced expression and purification of cloned recombinant CMPK, as described in Example 1 ;
  • Figure 1 1 A depicts the amplification of the coding region of E. coli AMPK in preparation for cloning
  • Figure 1 IB depicts the induced expression and purification of cloned recombinant AMPK, as described in Example 1 ;
  • Figure 12A depicts the amplification of the coding region of E. coli UMPK in preparation for cloning
  • Figure 12B depicts the induced expression and purification of cloned recombinant UMPK, as described in Example 1 ;
  • Figure 13 depicts the amplification of the coding region of E. coli TMPK in preparation for cloning into an expression vector, as described in Example 1 ;
  • Figure 14 shows the effect of GDP and GTP on the NER catalyzed by recombinant E. coli GMPK, as detected by the bioluminescence generated by a coupled luciferase assay, as described in Example 2;
  • Figure 15 depicts the results of an assay in which GDP is quantified using a NER catalyzed by recombinant E. coli GMPK, as detected by the bioluminescence generated by a coupled luciferase assay, as described in Example 3;
  • Figure 16 depicts the results of an assay in which the GTPase activity of DLP l is quantified using a NER catalyzed by recombinant E. coli GMPK in the manner show in Figure 7 (Note: K38A is an inactive point mutant form of DLPl , and WT is wildtype DLP l) , as described in Example 4;
  • Figure 17 shows the results of an exemplary high throughput screen quantifying DLP l GTPase activity using the GMPK-catalyzed NER depicted in Figure 16, as described in Example 5;
  • Figure 18A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by CMPK and driven with GTP (as the BTP), is used to detect and quantify CMP
  • Figure 18B shows exemplary results obtained with this embodiment of the invention, as described in Example 6;
  • Figure 19A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by AMPK and driven with dATP (as the BTP), is used to detect and quantify AMP
  • Figure 19B shows exemplary results obtained with this embodiment of the invention, as described in Example 7;
  • Figure 20A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by UMPK and driven with dATP (as the BTP), is used to detect and quantify UMP
  • Figure 20B shows exemplary results obtained with this embodiment of the invention, as described in Example 8.
  • the present invention provides a new approach for developing assays for the qualitative and quantitative analysis of biologically-relevant nucleotides other than ATP, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP.
  • the approach described herein can also be used to create assays that allow researchers to monitor the hydrolysis of nucleoside triphosphates by NTPases or dNTPases other than ATPase.
  • the present invention provides methods for the qualitative and quantitative analysis of biologically-relevant nucleotides other than ATP, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP. Also, using the approach disclosed, the present invention provides methods for monitoring the hydrolysis of nucleoside triphosphates by NTPases or dNTPases other than ATPase.
  • this approach is based upon a novel process comprising a first reaction coupled to a second reaction, wherein the first reaction is catalyzed by an NMPK and generates ATP, and the second reaction detects the ATP generated by said first reaction.
  • the present invention further provides a process wherein an NMPK acts to transfer a phosphate group from a phosphate donor to a phosphate acceptor and the NMPK acts to further transfer a phosphate group to ADP to generate ATP.
  • the present invention provides novel methods for the quantitative and qualitative analysis of a wide variety of nucleotides, other than ATP, and provides novel methods for quantifying their hydrolysis.
  • the assays of the present invention are applicable to a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research in which it is necessary to detect the presence of, or quantify the amount of, a particular nucleotide, or to quantify the activity of a particular NTPase. Because of their broad utility and ready adaptability of the assays disclosed, these assays are of value to the biochemist, molecular biologist and clinician, among others.
  • the methods and assays present invention can be adapted for use in screening assays, such as assays designed to detect the inhibition of particular NTPases, including GTPases.
  • the nexus of the disclosed methods and assays involves the coupling of an ATP-generating cyclic reaction known generally as a Nucleotide Exchange Reaction (or "NEPv”) to any suitable reaction that serves as a means of detecting the NER-generated ATP (i. e. , an "ATP detection reaction” or "ADR").
  • NEPv Nucleotide Exchange Reaction
  • ADR ATP detection reaction
  • Nucleoside Monophosphate Kinase (or NMPK) that acts to transfer a high-energy phosphate from a substrate ribonucleotide triphosphate (NTP) or, in some cases, a deoxyribonucleotide triphosphate (dNTP), to adenosine diphosphate (ADP), thereby generating the ATP that is detected by the coupled ATP detection reaction or ADR.
  • NTP ribonucleotide triphosphate
  • dNTP deoxyribonucleotide triphosphate
  • ADP adenosine diphosphate
  • the NER may be configured to generate ATP in an NTP- (or dNTP-), NDP- (or dNDP-) or NMP- (or dNMP-) dependent fashion.
  • assays according to the present invention can be configured to qualitatively or quantitatively assay for the presence of virtually any specific, biologically-relevant NTP, NDP, NMP, dNTP, dNDP, or dNMP, with the exception of ATP.
  • the reason that the reactions of the present invention cannot be used for the qualitative or quantitative analysis of ATP, is that the NER ultimately generates ATP, which is then detected and quantified by a coupled ADR.
  • the ATP generated by an NER can be detected by any suitable means or method of ATP detection that is specific for ATP, and that hydrolyzes or otherwise destroys or removes the ATP being detected.
  • One such ATP detection method involves the ATP-dependent generation of light by a second reaction catalyzed by an enzyme known as a "luciferase.” This method is specific for ATP because firefly luciferases specifically utilize ATP as a substrate in the reaction that generates light.
  • the present invention can also be used to quantify the amount of nucleotide hydrolysis (other than ATP hydrolysis) catalyzed by a particular enzyme, when the hydrolysis results in the production of a nucleoside diphosphate or monophosphate that can be used as a substrate by a particular NMPK.
  • the method works as follows: Nucleotide hydrolysis by the enzyme in under study generates a nucleotide substrate for a particular NMPK that, in turn, catalyzes an NER that generates ATP from ADP in a substrate-dependent fashion.
  • the ATP so-generated is then utilized by an ADR, such as a luciferase-catalyzed production of light, to quantify the amount of ATP produced by the NER.
  • the ADR must be specific for ATP, and for luciferase-catalyzed ADRs, this specificity results from the substrate specificity of luciferases, which utilize ATP, and no other nucleoside triphosphates, as a substrate in the reactions they catalyze to generate light. Under such conditions the nucleotide triphosphates (other than ATP) hydrolyzed by the enzyme under study do not contribute to the luminescent signal generated by luciferase, and thus, do not interfere with the "readout" portion of the assay.
  • the present invention provides reaction mixtures that may be used to conduct these assays.
  • the present invention also provides kits that may be used for conducting such assays.
  • NMPKs Nucleoside monophosphate kinases
  • this reaction involves the transfer of a high energy phosphate from ATP to a nucleoside
  • NMP nucleoside diphosphate
  • reverse reaction the reaction involves a transfer of a phosphate from NDP to ADP, thereby generating ATP and the corresponding NMP. It is the readily reversible nature of the reaction catalyzed by NMPKs that makes the approach and assays of the present invention possible.
  • the NMPK enzyme acts upon two substrates to form two products, and that the differences observed between the substrates and products involves the redistribution of a single phosphate group.
  • NMPKs fall into different subclasses that have evolved to catalyze the above reversible reaction using specific subsets of NMPs and NDPs. These subclasses of enzymes are named for the NMP/NDP substrates to which they add and remove phosphates by the phosphotransfer reactions they catalyze.
  • NMPKs that transfer phosphates from ATP to AMP in the forward reaction depicted above are known as adenosine monophosphate kinases, AMPKs, or adenylate kinases (AKs).
  • NMPKs that transfer phosphates from ATP to GMP in the forward reaction depicted above are known as guanosine monophosphate kinases, GMPKs, or guanylate kinases (GKs).
  • NMPKs that transfer phosphates from ATP to CMP in the forward reaction depicted above are known as cytidine monophosphate kinases, AMPKs, or cytidylate kinases (CKs).
  • NMPKs that transfer phosphates from ATP to TMP in the forward reaction depicted above are known as thymidine monophosphate kinases, TMPKs, or thymidylate kinases (TKs).
  • NMPKs that transfer phosphates from ATP to UMP in the forward reaction depicted above are known as uridine monophosphate kinases, UMPKs, or uridylate kinases (UKs). All of five of these subclasses of NMPKs have been classified by the International Union of Biochemistry and Molecular Biology (IUBMB) as members of enzyme class EC 2.7.4.4 and members of each subclass are found in all living organisms.
  • IUBMB International Union of Biochemistry and Molecular Biology
  • NMPKs generally follow a random Bi-Bi kinetic mechanism in catalyzing the above reaction in both directions.
  • Structural studies have revealed that each NMPK molecule has two separate binding sites for the two substrates; one for the phosphate donor nucleotide (the NTP site), and the phosphate acceptor nucleotide (the NMP site).
  • NTP site the phosphate donor nucleotide
  • NMP site the phosphate acceptor nucleotide
  • NMPKs are not very specific with respect to the phosphate donors they utilize. While most NMPKs prefer ATP at their NTP site (in some cases by an order of magnitude more than other NTPs), most NMPKs will utilize other NTPs, and even sometimes dNTPs, as phosphate donors for the forward reaction depicted above.
  • NMPKs In contrast to the NTP site, the NMP site of NMPKs has been found to make extensive interactions with the substrate NMP.
  • NMPKs will utilize a variety of phosphate donors other than ATP, they tend to be quite specific for a given subclass of phosphate acceptors.
  • AMPKs greatly prefer AMP as the phosphate acceptor in the forward reaction.
  • GMP GMP binding site
  • UMPKs appear to have the lowest specificity with respect to phosphate acceptors. They will readily catalyze phosphoryl transfer to CMP and AMP, in addition to UMP.
  • NMPKs While most NMPKs show a high degree of specificity for their cognate phosphate acceptors at their NMP site, several NMPKs have been found by the inventors to tolerate deoxyribose-containing NMPs at this site. This means that in certain situations, dNMPs can be used as phosphate acceptors in the forward reaction, while dNDPs can be used as a source of phosphate to be transferred to ADP (forming ATP) in the reverse reaction.
  • ADP forming ATP
  • CMPK from Escherishia coli will readily use GTP, instead of CTP as a phosphate donor.
  • AMPK, GMPK and UMPK also from E. coli, will effectively and readily utilize dATP as a phosphate donor, instead of their cognate respective phosphate donors, ATP, GTP and UTP.
  • This "relaxed specificity" of NMPKs for specific phosphate donors at their NTP site greatly aids in the design of assays for nucleotides other than ATP.
  • coli to tolerate deoxyribose-containing NMPs at their NMP sites, means that these assays can be adapted for the detection and quantification of a wide variety of dNTPs, dNDPs and dNMPs, in addition to NTPs (other than ATP), NDPs, and NMPs. The importance of this fact will be further revealed in the Examples described below.
  • NER Nucleotide Exchange Reaction
  • the phosphate on the diphosphorylated NDP or dNDP is transferred to ADP, thereby creating a molecule of ATP and "regenerating" the starting phosphate acceptor NMP or dNMP.
  • NMPK To catalyze both the forward and reverse reactions of an NER as depicted Figure 1 , the NMPK used must possess the appropriate substrate specificity required. Since, as discussed above, most NMPKs will utilize a variety of phosphate donors but generally tend to be quite specific for a given subclass of phosphate acceptors, the type of NMPK chosen to catalyze the NER will largely be determined by the subclass of phosphate acceptors to be detected or quantified. Hence, should one wish to assay for GMP, GDP or GTP, GMPK would generally be chosen as the NMPK to catalyze the NER.
  • the choice of subclass of NMPK to use for catalyzing the NER should generally match the subclass of phosphate acceptors used in the forward reaction (i.e. , GMPK for GMP, CMPK for CMP, etc.).
  • non-cognate BTPs can often be employed as the phosphate donor to "drive" the forward reaction by mass action. This fact, while subtle, is important, since it allows the forward reaction to be "driven” by a phosphate donor that, once converted to a diphosphate, will not be a favored substrate for the reverse reaction.
  • the examples below provide several embodiments of the assays of the present invention where a non-cognate phosphate donor is used to drive the forward reaction of the NER.
  • Figure 2 depicts how the ATP produced from one cycle of the NER can be used as an intermediary to couple the NER to a particular ATP detection reaction, or ADR. As depicted in Figure 2 the phosphate originally on the phosphate donor BTP is ultimately transferred to ADP to create the ATP that is detected by the "coupled" ADR.
  • the ADR employed releases the chemical energy stored in ATP to generate light, as shown.
  • the ATP produced by the NER is utilized by the enzyme luciferase, in the presence of the substrate luciferin and molecular oxygen (0 2 ) to generate light, AMP, PPi, Oxyluciferin and C0 2 .
  • the light produced by this reaction may be conveniently detected by any appropriate means, including photomultiplier tubes and other types of optical sensors, or by the exposure of photographic film. Since firefly luciferase only uses ATP as a substrate, and not other NTPs or dNTPs, other types of nucleoside triphosphates will not interfere with the ADR catalyzed by luciferase.
  • the ADR consumes the ATP generated by the NER, in this case converting it to AMP and PPi.
  • the AMP created by this ADR cannot be used as a substrate by the NMPK catalyzing the NER if the NMPK is not AMPK. If however, the NMPK being used is AMPK, the rate of the NER will still be limited by the quantity of BTP present.
  • the process involved must either consume the ATP created by the NER, or otherwise remove it from the reaction mixture, so as not to inhibit the NER by "product inhibition" that would result from increased concentrations of ATP.
  • Figure 3 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NMP or dNMP in a sample.
  • reaction mix comprising:
  • NMPK i.e. , an NMPK having a phosphate acceptor specificity
  • a BTP that can either be from the same class of nucleotide as the NMP or dNMP to be detected, or preferably is from a different class of nucleotides, and
  • the appropriate reagents of the chosen ADR should be included.
  • the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (0 2 ).
  • the coupled NER and ADR will be dependent upon the NMP or dNMP to be assayed.
  • NMP or dNMP when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed forward reaction to proceed, thereby generating BDP and NDP or dNDP.
  • the NDP or dNDP so-generated, will allow the NMPK-catalyzed reverse reaction to proceed, and a phosphate will be transferred from the NDP or dNDP, to the ADP, thereby generating ATP.
  • the ATP so-generated, will be utilized by the luciferase, along with the luciferin and 0 2 , to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C0 2 , and light.
  • the amount of light produced will ultimately be dependent upon the amount of NMP or dNMP added to the reaction mixture in the form of the added sample.
  • the sample to be added is not a purified sample free of "intrinsic ATP,” but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted” as background from the amount detected by the coupled NER and ADR.
  • Examples 6, 7, and 8 provide working examples of assays of the type described above, in which the presence and relative amounts of CMP, AMP, and UMP are, respectively, detected.
  • Figure 4 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NDP or dNDP in a sample. Note that in this depiction, as in this configuration, the NMPK is used exclusively to catalyze the reverse reaction; however, BTP can be included to facilitate amplification of the signal generated.
  • reaction mix comprising:
  • NMPK i.e. , an NMPK having a phosphate acceptor specificity
  • a BTP can be included, but is optional. If included, the BTP can either be from the same class of nucleotide as the NDP or dNDP to be detected, or preferably is from a different class of nucleotides. Inclusion of a BTP can serve to amplify the signal, since the NMP or dNMP generated by the reverse reaction will serve as a phosphate acceptor in a subsequent forward reaction wherein the phosphate is donated by BTP.
  • the appropriate reagents of the chosen ADR should be included.
  • the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (0 2 ).
  • the coupled NER and ADR will be dependent upon the NDP or dNDP to be assayed.
  • NDP or dNDP when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed reverse reaction to proceed, thereby generating ATP, and NMP or dNMP.
  • the ATP so- generated, will be utilized by the luciferase, along with the luciferin and 0 2 , to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C0 2 , and light.
  • the amount of light produced will ultimately be dependent upon the amount of NDP or dNDP added to the reaction mixture in the form of the added sample.
  • the sample to be added is not a purified sample free of "intrinsic ATP,” but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted” as background from the amount detected by the coupled NER and ADR.
  • Examples 3 , 7, and 8 (below) provide working examples of assays of the type described above, in which the presence and relative amounts of GDP, ADP, and UDP are, respectively, detected.
  • NTPs Other Than ATP i.e., BTPs
  • Figure 5 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NTP other than ATP (i. e. , BDP) in a sample.
  • a reaction mix must be assembled comprising:
  • cognate NMP or dNMP i. e. , matching the phosphate acceptor specificity of the NMPK to be used
  • the appropriate reagents of the chosen ADR should be included.
  • the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (0 2 ).
  • the coupled NER and ADR will be dependent upon the BTP to be assayed.
  • BTP when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed forward reaction to proceed, and will provide the phosphate to be transferred to the included NMP or dNMP.
  • the forward reaction will thereby generate BDP and NDP or dNDP.
  • the NDP or dNDP so- generated, will allow the NMPK-catalyzed reverse reaction to proceed, and a phosphate will be transferred from the NDP or dNDP, to the ADP, thereby generating ATP.
  • the ATP so-generated, will be utilized by the luciferase, along with the luciferin and 0 2 , to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C0 2 , and light.
  • the amount of light produced will ultimately be dependent upon the amount of BTP added to the reaction mixture in the form of the added sample.
  • Example 2 provides a working example of assays of the type described above, in which the presence and relative amounts of GTP (and GDP) are detected.
  • Figure 6 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular cyclic nucleotide in a sample.
  • the cyclic nucleotide i. e. , cNMP
  • cNMP cyclic nucleotide
  • the sample to be added is not a purified sample free of "intrinsic ATP,” but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted” as background from the amount detected by the coupled NER and ADR.
  • Figures 7 and 8 depict two variations on how the coupled NER and ADR of the invention may be configured to specifically quantify the amount of a NTP hydrolysis catalyzed by an NTPase present in a sample, for example, a GTPase.
  • the GTPase generates GDP, which is then used to drive the reverse reaction catalyzed by GMPK.
  • the GDP generated by the GTPase catalyzed hydrolysis is detected using the same approach depicted in Figure 4, and outlined in the section entitled Detection and Quantification of NDPs or dNDPs.
  • FIG 7 a generalized scheme is depicted in which a BTP other than ATP, and other than GTP is provided to amplify the signal generated.
  • a BTP other than ATP and other than GTP is provided to amplify the signal generated.
  • GTPases have a significantly higher affinity for GTP than does GMPK, which normally would utilize ATP as a phosphate donor, and GMP as a phosphate acceptor in the forward reaction. Inclusion of excess BTP, however, would effectively compete with GTP for use as a phosphate donor.
  • the specific BTP used is dATP, which can be used as a phosphate donor by GMPK, but is not used as a substrate by luciferase.
  • a BTP (such as dATP, as shown in Figure 8) can be included, but is optional. If included, the BTP is preferably from a different class of nucleotides than GTP (such as dATP), and is included in excess over GTP. However, as noted above, in most cases, the GTPase being assayed would have a significantly greater affinity for GTP than the GMPK. Also as noted, inclusion of a BTP serves to amplify the signal, since the GMP generated by the reverse reaction will serve as a phosphate acceptor in a subsequent forward reaction wherein the phosphate is donated by BTP.
  • GTP such as dATP
  • the appropriate reagents of the chosen ADR should be included.
  • the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (0 2 ).
  • the coupled NER and ADR will be dependent upon the GDP produced by the hydrolysis of GTP catalyzed by the GTPase.
  • GDP when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed reverse reaction to proceed, thereby generating ATP, and GMP.
  • the ATP so-generated, will be utilized by the luciferase, along with the luciferin and 0 2 , to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C0 2 , and light.
  • the amount of light produced will ultimately be dependent upon the amount of GDP produced by the GTPase in the added sample.
  • the sample to be added is not a purified sample free of "intrinsic ATP,” but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted” as background from the amount detected by the coupled NER and ADR.
  • Example 4 provides a working example of assays of the type described above, although no additional BTP or dATP was added, demonstrating the optional nature of the BTP or dATP addition.
  • the methods and assays present invention can be adapted for use in screening assays, such as assays designed to detect the inhibition of particular NTPases, including GTPases.
  • screening assays can be used to detect and study inhibitors of specific NTPases.
  • the present invention provides methods of screening test compounds for their ability to inhibit a particular GTPase present in a sample.
  • Such methods comprising combining said sample with a test compound, GTP, a purified guanosine monophosphate kinase (GMPK), ADP; and optionally, an NTP, other than ATP or GTP, capable of use as a phosphate donor by the GMPK, and detecting any ATP generated.
  • the amount of ATP generated is indicative of the amount or activity of the particular GTPase present in said sample, and a reduction of the ATP generated in the presence of said test compound, relative to the amount generated in the absence of said test compound, indicates that said test compound inhibits said particular GTPase.
  • GTP phosphoglycerate kinase
  • the present invention provides reaction mixtures designed for practicing the disclosed methods and assays. These reaction mixtures can be configured differently, depending upon the specific assay to be conducted, however, the reaction mixtures must eventually include a purified recombinant NMPK in an appropriate buffer at an appropriate concentration, for catalyzing a particular NER required for conducting an assay of the invention.
  • the purified recombinant NMPK is chosen from AMPK, CMPK, GMPK, TMPK and UMPK according to nucleotide that is to be qualitatively or quantitatively analyzed, or the substrate requirements of the NTPase whose activity is to be assayed.
  • reaction mixtures may also contain Mg 2+ in a
  • reaction mixtures may also contain or one or more sufficiently pure nucleotides (i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate) to serve as a substrate (i. e. , phosphate acceptor or phosphate donor) as required for the NER to be catalyzed by the selected purified recombinant NMPK.
  • nucleotides i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate
  • substrate i. e. , phosphate acceptor or phosphate donor
  • reaction mixture comprising:
  • NMPK i.e. , an NMPK having a phosphate acceptor specificity
  • a BTP that can either be from the same class of nucleotide as the NMP or dNMP to be detected, or preferably is from a different class of nucleotides, and
  • reaction mixture comprising:
  • NMPK i.e. , an NMPK having a phosphate acceptor specificity
  • reaction mixture comprising:
  • cognate NMP or dNMP i. e. , matching the phosphate acceptor specificity of the NMPK to be used
  • reaction mix comprising:
  • NTPase i.e., GMPK for GDP
  • these reaction mixtures may also optionally contain Mg 2+ , in a concentration appropriate for use as a required cofactor of the included NMPK.
  • the reaction mixtures may optionally contain or one or more sufficiently pure additional nucleotides (i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate) to serve as a substrate (i. e. , phosphate acceptor or phosphate donor) as required for the NER to be catalyzed by the included purified recombinant NMPK, or as required to amplify the signal (i. e. , ATP) generated by the NER through subsequent cycles of the NER.
  • additional nucleotides i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate
  • the reaction mixtures may also optionally contain those reagents required for the ADR that is to be coupled to the NER.
  • Such reagents may include, for example, luciferase enzyme and luciferin.
  • kits designed to facilitate practicing the disclosed methods and assays. These kits can be configured in various ways, containing various sets of reagents, depending upon the specific method or assay to be conducted, or, alternatively, they can include a collection of reagents required to conduct a plurality of the methods and assays of the invention. Ideally the kits include a purified recombinant NMPK at an appropriate concentration, and in an appropriate buffer in a suitable resealable container, and (2) instructions for the use of the purified
  • NMPK recombinant NMPK to conduct a method or methods, or an assay or assays, of the invention, which have as a nexus the NER catalyzed by the included purified
  • NMPK recombinant NMPK.
  • the NMPK is chosen from AMPK, CMPK, GMPK, TMPK and UMPK, depending upon the methods and assays that are to be conducted.
  • kits may also contain Mg 2+ in water or in a suitable buffer, at a concentration appropriate for use as a required cofactor of the included NMPK, in a suitable container.
  • the kits may also contain, in suitable containers, one or more solutions comprising a sufficiently pure nucleotide (i. e. , nucleoside triphosphate, nucleoside diphosphate or nucleoside monophosphate) for use either in either conducting a method or assay of the invention (i. e. , which are based upon the NER catalyzed by the included purified recombinant NMPK), or for use as a positive or negative control sample.
  • a sufficiently pure nucleotide i. e. , nucleoside triphosphate, nucleoside diphosphate or nucleoside monophosphate
  • the one or more sufficiently pure nucleotides may be provided in a dried or lyophilized form that can be rehydrated, resuspended or dissolved by the end user to form a solution, or solutions, for use in the methods and assays of the invention.
  • kits may also contain one or more suitable containers containing a solution comprising a cyclic nucleotide phosphodiesterase, for use in assays designed to detect or quantify a particular cyclic nucleotide in a sample, as shown in Figure 6.
  • the kits may also contain one or more suitable containers containing a solution comprising a GTPase to be used as a positive control in assays of GTPase activity, such as those depicted in Figures 7 and 8, or described in Example 4 or 5, below.
  • kits of the invention also contain reagents sufficient for conducting the ADR, which is to be coupled to the NMPK-catalyzed NER that the kit is designed to facilitate.
  • any suitable ADR can be utilized for the assays of the present invention, so long as the ADR can be coupled to the NER, and so long as the ADR consumes or otherwise eliminates the ATP generated by the NER.
  • the ADR involves the generation of light by the reaction catalyzed by ATP-dependent luciferase.
  • the kits of the present invention can comprise the reagents required to conduct the ADR, including purified recombinant luciferase and luciferin.
  • One particular set of embodiments involve the use of firefly luciferase, and its substrates, luciferin and 0 2 , in the ADR to detect the ATP generated by an NER of the present invention.
  • One commercial embodiment along these lines is the
  • ATPLiteTM system which is available from PerkinElmer of Waltham, MA. Details of the specific reagents and reactions at the core of the ATPLiteTM system are found in U.S. Patent 6,503,723, the contents of which are incorporated by reference herein in their entirety.
  • kits of the present invention particularly comprise and contain the reagents of the PerkinElmer ATPLiteTM system, or the reagents taught in U.S. Patent 6,503,723.
  • the kits of the present invention contain some or all of the reagents required to conduct a particular methods or assay of the invention in some type of carrier or compartmentalized container.
  • the kits of the invention contain some or all of the reagents required to conduct a particular NER and coupled ADR, in some type of carrier or compartmentalized container.
  • the reagents included in the kits are contained within appropriate resealable containers. Ideally such containers are included in the kits in specific locations of the carrier or compartmentalized container.
  • the choice of specific location for a give reagent should ideally create some degree of organization or orchestrated grouping of the reagents. For example, all of the reagents required for the NER may be grouped in one group, while the reagents required for the ADR may be grouped in a second group.
  • the carrier or compartmentalized container can comprise a container, vessel, or support, in the form of, e.g. , a bag, box, foam block, or rack, that is optionally compartmentalized.
  • the carrier may define an enclosed confinement for safety purposes during shipment and storage, and such a carrier may be designed to enclose the compartmentalized container.
  • the carrier or compartmentalized container may ideally be designed such that the components carried therein remain in an organized state during shipment and storage.
  • the carrier optionally contains instructions for the use of the contents of the carrier in conducting one or more of the methods and assays of the present invention.
  • Example 1 Cloning, Overexpression & Purification of AMPK, CMPK, GMPK and
  • the Escherischia coli K- 12 strain, MG1655 ⁇ i. e. , DH5 ) substrain possesses five different NMPK-encoding genes. These genes have been given different names as a result of multiple naming conventions.
  • the table below provides: (a) the names of the different NMPK genes associated with the NMPK they encode; (b) the EcoGene Accession Numbers for these genes (as provided by the EcoGene database maintained by the University of Miami and available via the internet at
  • Coli K-12 strain DH10B or DH5a cells were heat treated to obtain a simple lysate, and this lysate was used as the source of template genomic DNA from which the NMPK coding regions were amplified using the different NMPK-specific primer pairs.
  • the primer pairs were designed to facilitate the specific amplification of a particular NMPK coding region, and were also designed to facilitate cloning of the amplified DNA.
  • Amplified DNAs i. e. , PCR products
  • NMPKs GMPK
  • CMPK CMPK
  • AMPK AMPK
  • UMPK UMPK
  • TMPK TMPK
  • Figs. 9A, 10A, 1 1A, 12A, and 13 gel purified
  • cloned, in-frame into a modified pET expression vector that included an upstream initiation codon followed by six in-frame histidine codons, under the control of a T7 promoter.
  • the completed expression vectors comprised a translation initiation codon encoding the N-terminal methionine, followed by six in-frame histidine codons that were, in-turn, positioned in frame with the native initiation methionine and subsequent coding region for the particular NMPK.
  • the six in-frame histidine codons served to add an N-terminal "hexa-histidine tag" (i.e. , "His6 tag”) to the expressed protein. This His6 tag facilitated rapid purification of recombinant protein as described below.
  • DNA sequencing was used to confirm that the sequence encoding the NMPK was properly inserted in-frame with the N-terminal His6 tag to create the desired expression vector.
  • the completed expression vectors were transformed into either the BL21 DE3 or BL21 AI strain, and expression of the encoded recombinant NMPK with N- terminal His6 tag, was induced with either IPTG for the transformed BL21 DE3 strain, or IPTG and L-arabinose for the transformed BL21 -AI strain. Cell densities and induction times required for maximal expression were determined on small batches of transformed cells, and expressions for purification were scaled up accordingly.
  • Transformed expression strains were grown to appropriate densities and induced to overexpress a particular recombinant His-tagged NMPK, for a desired period of time.
  • Cells were isolated by centrifugation and lysed. Lysates were applied to Ni 2+ - agarose columns in order to isolate the recombinant His-tagged NMPK by immobilized metal ion affinity chromatography.
  • the His6-tagged, recombinant NMPKs adhered to the column, while the majority of other cellular proteins eluted from the column.
  • the bound His-tagged, recombinant NMPKs were eluted from the column using a gradient of increasing concentrations of imidizole (Figs. 9B, 10B, 1 1B, & 12B). Isolated fractions were collected and analyzed by SDS-PAGE (Figs. 9B & 10B).
  • His-tagged recombinant E. Coli NMPKs were isolated to high levels of purity ⁇ i. e. , 90% or greater), and yields from about 100 to 400 mg of recombinant protein per liter of cultured transformed E. coli were obtained.
  • Representative amplification steps for GMPK, CMPK, AMPK, UMPK, and TMPK are show in Figures 9A, 10A, 1 1A, 12A, and 13, respectively.
  • Representative purification steps for GMPK, CMPK, AMPK, and UMPK are show in Figures 9B, 10B, 1 1B, and 12B, respectively.
  • E. Coli NMPKs ⁇ i. e. , His6-GMPK, His6-CMPK, His6- AMPK, and His6-UMPK
  • His6-GMPK His6-GMPK
  • His6-CMPK His6-CMPK
  • His6-AMPK His6-AMPK
  • His6-UMPK His6-UMPK
  • a GMPK-catalyzed NER was coupled with a luciferase-catalyzed ATP detection reaction (ADR) as schematically shown in Figure 2.
  • ADR luciferase-catalyzed ATP detection reaction
  • the basic reactions were set up as follows: A 100 ⁇ total volume sample (containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 10 ⁇ ADP, +/- 10 ⁇ GTP, +/- 0.1 ⁇ GMPK) is titrated with increasing concentrations of GDP from 0.005 ⁇ to 5 ⁇ . The reaction was incubated at room temperature for 30 min. whereupon 50 ⁇ of ADR solution (ATPLiteTM solution; PerkinElmer; Waltham, MA) was added to the samples and the luminescent signal measured with a Topcount plate reader (PerkinElmer; Waltham, MA).
  • ADR solution ATPLiteTM solution
  • PerkinElmer Waltham, MA
  • the reaction mixture contained 10 mM Mg 2+ , 10 ⁇ ADP, 10 ⁇ GTP, 0.1 ⁇ GMPK, and increasing concentrations of GDP from 0.005 to 5 ⁇ . Control reactions were conducted in which GTP, GMPK, or both, were left out. The results are shown in Figure 14. [00127] It should be noted that, at sufficient levels, GDP can be readily detected and quantified when the same reaction is conducted in the absence of GTP. However, addition of GTP to the reaction mixture results in the amplification of the signal generated, due to the cyclic nature of the NER. This assay was found to be highly sensitive and can be used to determine concentrations of GDP as low as 0.05 ⁇ .
  • DLP 1 also known as dynamin 1 -like; dynamin-like protein 4; dynamin- like protein IV; Dnmlp/Vpslp-like protein; and dynamin-related protein 1 ; and encoded by Entrez GenelD: 10059
  • dynamin 1 is a member of the dynamin superfamily of GTPases.
  • dynamin-related subfamily including the S. cerevisiae proteins Dnml and Vps l , contain the N-terminal tripartite GTPase domain but do not have the pleckstrin homology or proline-rich domains. This protein establishes mitochondrial morphology through a role in distributing mitochondrial tubules throughout the cytoplasm.
  • the methods of the present invention were utilized to develop an assay for the GTPase activity of recombinant DLP l .
  • the assay utilized a GMPK-catalyzed NER coupled with a luciferase-catalyzed ADR to detect the GDP produced as a result of GTP hydrolysis catalyzed by DLP l , as shown schematically in Figure 7.
  • the activity of wild type DLPl was compared to that of a catalytically-deactivated mutant form, in which a key lysine residue (K38) was substituted with an alanine. This "K38A" mutant form of DLP l was used a negative control.
  • Fig. 16 Experiments using a wild type DLP l and catalytically inactive K38A DLP l are shown in Fig. 16.
  • the 75 ⁇ total volume samples contained 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 10 ⁇ GTP, and 0.2-1 ⁇ DLP l WT or DLP (K38A) mutant protein as indicated. Reactions were performed at room temperature for 50 minutes, whereupon ADP and GMPK were added to final a concentration of 15 ⁇ and 0.1 ⁇ , respectively. Samples were incubated at room temperature for 10 min. and 50 ⁇ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was then added to the samples. Luminescence signal was measured with a Topcount plate reader
  • Figure 16 shows the results of the assay conducted with three different concentrations of wild type or K38A DLP.
  • Increasing concentrations (0.2, 0.5 and 1.0 ⁇ ) of wild type DLPl resulted in a substantial increase in the amount of light generated.
  • increases in concentrations (0.2, 0.5 and 1.0 ⁇ ) of the K38A mutant form of DLP l resulted in a slight increase in the amount of light generated. This slight increase in light may be due to the K38A form of DLPl having a very small amount of catalytic activity, relative to the wild type enzyme.
  • GMPK-catalyzed nucleotide exchange reactions can be used for high throughput screening of compounds which inhibit DLPl enzymatic activity.
  • Solution compositions to be used are as indicated below.
  • Mixing 30 ⁇ Solution A and 10 ⁇ Solution B results in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 0.01 % (w/v) Triton X- 100, 10 ⁇ ADP, 10 ⁇ GTP, and 0.5 ⁇ DLP l .
  • 0.5 ⁇ of compound or vehicle alone was then added to the samples and incubated at room temperature for 1 - 1.5 hours.
  • Triton X- 100 Can be stored w/o Triton X- 100 at 4°C for up to a week; add Triton X- 100 immediately before use.
  • the reaction mixture contains Tris buffer, 10 mM MgCl 2 , 10 ⁇ ADP, 10 ⁇ GTP, 0.1 ⁇ GMPK. 0.4 ⁇ wild type DLP l (upper set of data points). Omitting DLP l from the mixture is designed as background (lower set of data points). ATP generated from the reaction is measured with commercially available luciferase based ATPlite solution (PerkinElmer, Cat # 6016949; Waltham, MA).
  • This standard HTS performance evaluation assay indicates that the NER based DLP l GTPase activity assay has excellent HTS performance quality (Z' factor) and excellent signal background and signal:noise ratios.
  • Example 6 Detection and Quantification of CMP Using CMPK-Catalyzed
  • the basic reactions were set up as follows: 100 ⁇ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 and the components indicated in Figure 18 (the numbers along the x axis indicate the concentrations of nucleotides, in ⁇ ). 0.1 ⁇ CMPK was present or absent as indicated. Samples were incubated at room temperature for 30 min, whereupon 50 ⁇ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was added and the luminescent signal measured with a Topcount plate reader.
  • ADR solution ATPLite solution; PerkinElmer; Waltham, MA
  • the phosphate donor employed in the forward reaction of the NER was GTP.
  • the results (Figure 18B) clearly demonstrate that the assay is CMP-dependent, and show that the use of GTP as the phosphate donor in the forward reaction works well, with only a slight background signal being generated.
  • the basic reactions were set up as follows: 80 ⁇ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgC12 and the components indicated in Figure 19 (the numbers along the x axis indicate the concentrations of nucleotides, in ⁇ ). 0.34 ⁇ AMPK was present or absent as indicated. Samples were incubated at room temperature for 60 min, whereupon 40 ⁇ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was added and the luminescent signal was measured with a Topcount plate reader (PerkinElmer; Waltham, MA).
  • ADR solution ATPLite solution
  • PerkinElmer Waltham, MA
  • AMPK can catalyze AMP phosphorylation with dATP as a phosphate donor, to produce ADP.
  • dATP a phosphate donor
  • no exogenous ADP is needed for the AMPK-catalyzed NER in order to detect and quantify levels of AMP.
  • one molecule of ADP can act as a phosphate donor for the AMPK-catalyzed phosphorylation of another molecule of ADP, thereby generating ATP and AMP.
  • the ATP generated by this reaction can then be detected by a coupled ADR, while the AMP can be reused for the cycling reaction, thereby amplifying the signal produced.
  • an AMPK-catalyzed NER coupled to a luciferase-catalyzed ADR was used to detect the presence of AMP alone, or ADP alone, in a sample.
  • the phosphate donor employed in the forward reaction of the NER was dATP.
  • the use of dATP, rather than ATP (or GTP), is made possible by the relatively relaxed substrate specificity of E. coli AMPK for the phosphate-donating nucleotide.
  • dATP is not a substrate for the luciferase-catalyzed ADR, and results in only a small background signal.
  • the basic reactions were set up as follows: 80 ⁇ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgC12 and the components indicated in Figure 20(the numbers along the x axis indicate the concentrations of nucleotides, in ⁇ ). 0.26 ⁇ UMPK was present or absent as indicated. Samples were incubated at room temperature for 60 min, whereupon 40 ⁇ of ADR solution (ATPLite solution) was added and the luminescent signal was measured with a Topcount plate reader.
  • ADR solution ATPLite solution
  • UMPK can catalyze UMP phosphorylation using dATP as a phosphate donor, to produce UDP.
  • UDP can act as a phosphate donor for the UMPK-catalyzed phosphorylation of exogenous ADP, to generate ATP and UMP.
  • the ATP generated by this reaction can then be detected by a coupled ADR, while the UMP can be reused for the cycling reaction, thereby amplifying the signal produced.
  • a UMPK-catalyzed NER coupled to a luciferase-catalyzed ADR was used to detect the presence of UMP alone, or UDP alone, in a sample.
  • the phosphate donor employed in the forward reaction of the NER was dATP.
  • the use of dATP, rather than UTP (or GTP), is made possible by the relatively relaxed substrate specificity of E. coli UMPK for the phosphate-donating nucleotide.
  • dATP is not a substrate for the luciferase-catalyzed ADR, and results in a very small background signal.

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Abstract

The present invention provides novel methods, reaction mixtures, and kits for the quantitative and qualitative analysis of nucleotides other than ATP, for the quantification of the hydrolysis of nucleotides other than ATP, and for the quantitative and qualitative analysis of the activity of enzymes that generate or consume nucleotides other than ATP.

Description

COUPLED REACTIONS FOR ANALYSIS OF NUCLEOTIDES
AND THEIR HYDROLYSIS
RELATED APPLICATIONS
[0001 ] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/255 ,068, filed October 26, 2009, the contents of which are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to biochemical assays, and particularly to methods for the quantitative and qualitative analysis of nucleotides and their hydrolysis.
BACKGROUND OF THE INVENTION
[0003] Nucleotides play many roles in living cells. In addition to their somewhat mundane role as the building blocks of the nucleic acids, nucleotides play vital and central roles in cellular metabolism and bioenergetics. Nucleotides (particularly nucleoside triphosphates, or NTPs) serve as the "energy currency" of the cell. In their high-energy phosphorylated forms, nucleoside triphosphates provide a source chemical energy for numerous metabolic transactions and transformations. Nucleotides (and nucleosides) also serve as structural and functional components of a variety of enzyme cofactors, as well as metabolic intermediates in a wide variety of enzymatic reactions. Finally, nucleotides (and nucleosides) serve as small-molecule messengers that can activate or facilitate the response of cells to hormones and a variety of extracellular stimuli.
[0004] As a consequence of the many important roles played by nucleotides in living systems, biochemists often desire to determine the types and amounts of nucleotides present both in vivo and in vitro, and measure their rates and extent of hydrolysis. In order to study various enzymatic reactions involving nucleotides, biochemists require simple and effective assays that allow for quantification of specific nucleotides, particularly just before, or immediately after, an enzymatic reaction occurs. Additionally, biochemists require assays that are specific for particular nucleotides that are involved in cellular signaling.
[0005] To date, the only convenient and commercially available nucleotide hydrolysis assays are designed to measure adenosine triphosphate (i. e. , ATP)
hydrolysis. Assays for analyzing the hydrolysis of other nucleotide triphosphates are limited by high background caused by acidic reaction conditions and a lack of sensitivity (>10 μΜ detection capability). Indeed, the commonly used assay for nucleotide hydrolysis uses a malachite green based colorimetric assay. (See, e.g. :
Kirchgesser & Dahlmann; A colorimetric assay for the determination of acid nucleoside triphosphatase activity; J. Clin. Chem. Clin. Biochem. 28 :407-41 1 , 1990.) In these assays, nucleotide hydrolysis releases inorganic phosphate (Pi). The Pi forms a yellowish complex with malachite green dye under highly acidic conditions. Because the assays are based upon changes in the optical density readout at a particular wavelength, the sensitivity of this assay type is relatively low (above 10 μΜ Pi).
Additionally, this type of assay requires the use of sulfuric acid for color generation. Sulfuric acid itself can cause spontaneous acid hydrolysis of nucleotides, thereby interfering with the assays by increasing background. Further, such acidic assay conditions are unsuitable for high-throughput screening (HTS) machineries.
[0006] In view of the above, there is a need for improved methods and assays that can be used for the quantitative and qualitative analysis of specific types of nucleotides, including nucleoside monophosphates, nucleoside diphosphates, nucleoside
triphosphates (other than ATP), and cyclic nucleotides, and there is a need for improved methods and assays with which to measure the hydrolysis of nucleotides (other than ATP) by enzymes that utilize such nucleotides.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides a new approach and methods for
developing assays for the qualitative and quantitative analysis of most biologically- relevant nucleotides other than ATP, as well as assays to monitor the hydrolysis of such nucleotides. Disclosed herein are working examples of specific assays for the qualitative and quantitative analysis of specific nucleotides and their hydrolysis that utilize this approach and these methods. Indeed, the inventive approach and methods described herein may be used to create a variety of assays that can be used to monitor a variety of enzymatic nucleotide hydrolysis reactions, and may be adapted to
qualitatively and quantitatively assay most biologically-relevant ribonucleotides and deoxyribonucleotides, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP. Thus the invention provides methods for the quantitative and qualitative analysis of a wide variety of nucleotides, other than ATP, and provides methods for quantifying their hydrolysis.
[0008] The methods and assays of the present invention are applicable to a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research. As a result of their ready adaptability and broad utility, these assays are of value to biochemists, molecular biologists and clinicians, among others.
[0009] The nexus of these methods and assays involves the coupling of an ATP- generating cyclic reaction referred to herein as a "Nucleotide Exchange Reaction" (or "NEPv") to any suitable reaction that serves as a means of detecting the ATP generated by the NER (i. e. , the "ATP detection reaction" or "ADR"). The NER comprises a reversible reaction catalyzed by a single enzyme. That enzyme is a Nucleoside
Monophosphate Kinase (or NMPK), which, in one direction (the "forward" reaction), acts to transfer a high-energy phosphate from a substrate ribonucleoside triphosphate (NTP) other than ATP, or in some cases, a deoxyribonucleoside triphosphate (dNTP), to a ribonucleoside monophosphate (NMP) or dexoyribonucleoside monophosphate (dNMP), to form two corresponding nucleoside diphosphates (NDP or dNDP). In the opposite direction (the "reverse" reaction), the NMPK catalyzes the transfer of a phosphate group from a ribonucleoside diphosphate (NDP), or in some cases a deoxyribonucleoside diphosphate (dNDP), to ADP to form ATP and a corresponding NMP or dNMP. With each "cycle" of the cyclic NER, a single ATP is produced, which then detected by the coupled ATP detection reaction or ADR. [0010] The NMPKs are a family of enzymes, the members of which have evolved to transfer phosphates between particular ribonucleotide triphosphates (NTPs) or, in some cases, a deoxyribonucleotidetriphosphates (dNTPs), and ADP. Hence, by the selective inclusion of a particular NMPK, and a particular set of substrates, the NER can be configured to generate ATP in an NTP- (or dNTP-), NDP- (or dNDP-) or NMP- (or dNMP-) dependent fashion, where the NTP, NDP or NMP (or dNTP, dNDP or dNMP) are members of a particular cognate family of nucleotides that can serve as substrates for the NMPK being used. For example, when GMPK is used to catalyze the NER, the NER can be configured such that the generation of ATP is dependent upon amount of GTP, GDP or GMP present. In other words, by altering which substrates are present, and which particular NMPK enzyme is used as a catalyst, the assay methods provided may be configured to qualitatively or quantitatively assay for the presence of specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, with the exception of ATP. The reason that ATP cannot be specifically detected or quantified by the methods provided is that ATP is the particular nucleoside triphosphate generated by the NER, which is then detected and quantified by the ADR.
[0011] It should be noted that the ATP generated by an NER can be detected by any suitable ATP detection method, so long as the ATP is either converted to ADP + inorganic phosphate (Pi) or AMP and pyrophosphate (PPi), or is otherwise removed from, or more generally consumed by the detection reaction (ADR). In most
embodiments the ATP detection method is a coupled enzymatic reaction that
specifically utilizes ATP as a substrate. One such ATP detection method (ADR) involves the ATP-dependent generation of light in a reaction catalyzed by a "luciferase" enzyme, such as that originally isolated from the firefly, Photinus pyralis. This second reaction is specific for ATP because firefly luciferases specifically utilize ATP, and no other nucleoside triphosphates, as a substrate in the reaction that generates light.
[0012] U.S. patents 5,583,024; 5,674,713 and 5,700,673 provide methods for the recombinant expression of luciferase derived from bioluminescent insects, specifically bioluminescent beetles of the order Coleoptera. U.S. patent 6,503,723 provides methods for detecting ATP in a sample using reactions catalyzed by luciferases that generate light, wherein the amount of light generated is indicative of the presence or absence, and the quantity, of ATP in the sample. The teachings of these four U.S.
patents (i. e. , 5,583,024; 5,674,713; 5,700,673; and 6,503,723) regarding ATP detection methods and ATP detection reactions are incorporated by reference herein in their entirety.
[0013] As will be shown, in addition to the qualitative and quantitative analysis of specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, the present invention can also be used to quantify the amount of nucleotide hydrolysis (other than ATP
hydrolysis) catalyzed by a particular enzyme, such as an NTPase, when the hydrolysis results in the production of a nucleoside diphosphate or monophosphate that can be used as a substrate by a particular NMPK. The method works as follows: Nucleotide hydrolysis catalyzed by an enzyme under study (i. e. , an NTPase) generates a substrate for a particular matched NMPK (i. e. , an NMPK that can use that substrate) that, in turn, catalyzes an NER that generates ATP from ADP in a substrate-dependent fashion. The ATP so-generated is then utilized by luciferase to produce a bioluminescent signal and AMP + pyrophosphate (PPi). As noted above, since firefly luciferases only utilize ATP as their high-energy substrate, other starting nucleoside triphosphates do not contribute to the luminescent signal generated by luciferase, and thus, do not interfere with the "readout" of the coupled assays.
[0014] The disclosed NER-based methods have been successfully applied to determine the presence and relative amounts of GTP, GDP, CMP, ADP, AMP, UDP and UMP, but can be used for the qualitative and quantitative analysis of many other types of NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs. The disclosed NER-based methods have also been used to develop an assay to measure the GTPase activity of a particular GTPase, and this assay has been successfully employed in a high-throughput screening (HTS) format. However, the disclosed NER-based methods can readily be applied to the study of other GTPases, and any other enzyme that catalyze reactions which generate specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, other than ATP. The disclosed NER-based methods can also be used for the qualitative and quantitative analysis of specific cyclic nucleotides (i. e. , cAMP, etc.). Assays based upon the methods of the present invention have been found to be convenient, sensitive, robust and economical. [0015] As will be seen from the examples provided, the disclosed NER-based assays may be used for the detection and quatification of a wide range of biologically- relevant nucleotides (other than ATP), and their hydrolysis. The disclosed NER-based assays may be advantageously employed in a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research.
[0016] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0017] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 schematically depicts a cyclic Nucleotide Exchange Reaction (NER) catalyzed by an NMPK, wherein a phosphate group is ultimately transferred from "BTP" (top left; wherein BTP refers to any suitable ribonucleoside triphosphate other than ATP, or any suitable deoxyribonucleoside triphosphate, including dATP) to ADP, to generate ATP (bottom left);
[0019] Figure 2 shows how an NER may be coupled with an ATP detection reaction (ADR) to create assays for the detection and quantification of a wide variety of nucleoside mono-, di-, and triphosphates;
[0020] Figure 3 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NMPs or dNMPs;
[0021 ] Figure 4 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NDPs or dNDPs;
[0022] Figure 5 depicts how the coupled reactions of Figure 2 may be used for the detection and quantification of NTPs, other than ATP, and dNTPs (i.e. , "BTPs"); [0023] Figure 6 shows how the coupled reactions of Figure 2 may be used for the detection and quantification of cyclic nucleotides, when further coupled with a decyclization reaction catalyzed by a cyclic nucleotide phosphodiesterase;
[0024] Figure 7 depicts one configuration of the coupled reactions of Figure 2 being used for the quantification of the activity of a GTPase, particularly when the GTPase has a substantially greater affinity for GTP than the GMPK being used to catalyze the NER;
[0025] Figure 8 depicts another configuration of the coupled reactions of Figure 2 being used for the quantification of the activity of a GTPase, particularly by employing an excess of dATP as a substrate for the GMPK being used to catalyze the NER (Note: dATP is not a substrate for firefly luciferase);
[0026] Figure 9A depicts the amplification of the coding region of E. coli GMPK in preparation for cloning, and Figure 9B depicts the induced expression and
purification of cloned recombinant GMPK, as described in Example 1 ;
[0027] Figure 10A depicts the amplification of the coding region of E. coli CMPK in preparation for cloning, and Figure 10B depicts the induced expression and purification of cloned recombinant CMPK, as described in Example 1 ;
[0028] Figure 1 1 A depicts the amplification of the coding region of E. coli AMPK in preparation for cloning, and Figure 1 IB depicts the induced expression and purification of cloned recombinant AMPK, as described in Example 1 ;
[0029] Figure 12A depicts the amplification of the coding region of E. coli UMPK in preparation for cloning, and Figure 12B depicts the induced expression and purification of cloned recombinant UMPK, as described in Example 1 ;
[0030] Figure 13 depicts the amplification of the coding region of E. coli TMPK in preparation for cloning into an expression vector, as described in Example 1 ;
[0031] Figure 14 shows the effect of GDP and GTP on the NER catalyzed by recombinant E. coli GMPK, as detected by the bioluminescence generated by a coupled luciferase assay, as described in Example 2;
[0032] Figure 15 depicts the results of an assay in which GDP is quantified using a NER catalyzed by recombinant E. coli GMPK, as detected by the bioluminescence generated by a coupled luciferase assay, as described in Example 3; [0033] Figure 16 depicts the results of an assay in which the GTPase activity of DLP l is quantified using a NER catalyzed by recombinant E. coli GMPK in the manner show in Figure 7 (Note: K38A is an inactive point mutant form of DLPl , and WT is wildtype DLP l) , as described in Example 4;
[0034] Figure 17 shows the results of an exemplary high throughput screen quantifying DLP l GTPase activity using the GMPK-catalyzed NER depicted in Figure 16, as described in Example 5;
[0035] Figure 18A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by CMPK and driven with GTP (as the BTP), is used to detect and quantify CMP, and Figure 18B shows exemplary results obtained with this embodiment of the invention, as described in Example 6;
[0036] Figure 19A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by AMPK and driven with dATP (as the BTP), is used to detect and quantify AMP, and Figure 19B shows exemplary results obtained with this embodiment of the invention, as described in Example 7; and
[0037] Figure 20A depicts an embodiment of the coupled reactions of Figure 2 in which an NER catalyzed by UMPK and driven with dATP (as the BTP), is used to detect and quantify UMP, and Figure 20B shows exemplary results obtained with this embodiment of the invention, as described in Example 8.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides a new approach for developing assays for the qualitative and quantitative analysis of biologically-relevant nucleotides other than ATP, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP. The approach described herein can also be used to create assays that allow researchers to monitor the hydrolysis of nucleoside triphosphates by NTPases or dNTPases other than ATPase.
[0039] Using the approach disclosed, the present invention provides methods for the qualitative and quantitative analysis of biologically-relevant nucleotides other than ATP, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP. Also, using the approach disclosed, the present invention provides methods for monitoring the hydrolysis of nucleoside triphosphates by NTPases or dNTPases other than ATPase.
[0040] In general terms, this approach is based upon a novel process comprising a first reaction coupled to a second reaction, wherein the first reaction is catalyzed by an NMPK and generates ATP, and the second reaction detects the ATP generated by said first reaction. The present invention further provides a process wherein an NMPK acts to transfer a phosphate group from a phosphate donor to a phosphate acceptor and the NMPK acts to further transfer a phosphate group to ADP to generate ATP.
[0041] Disclosed herein are working examples of specific assays designed using this approach for the quantitative and qualitative analysis of GTP, GDP, CMP, AMP, ADP, UMP and UDP. Also disclosed is a working example of an assay also designed using this approach that may be used to quantify the activity virtually any GTPase, and that may be used in a high-throughput format.
[0042] Thus the present invention provides novel methods for the quantitative and qualitative analysis of a wide variety of nucleotides, other than ATP, and provides novel methods for quantifying their hydrolysis.
[0043] The assays of the present invention, and assays developed using the disclosed inventive approach, are applicable to a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research in which it is necessary to detect the presence of, or quantify the amount of, a particular nucleotide, or to quantify the activity of a particular NTPase. Because of their broad utility and ready adaptability of the assays disclosed, these assays are of value to the biochemist, molecular biologist and clinician, among others.
[0044] Additionally, the methods and assays present invention can be adapted for use in screening assays, such as assays designed to detect the inhibition of particular NTPases, including GTPases.
[0045] The nexus of the disclosed methods and assays involves the coupling of an ATP-generating cyclic reaction known generally as a Nucleotide Exchange Reaction (or "NEPv") to any suitable reaction that serves as a means of detecting the NER-generated ATP (i. e. , an "ATP detection reaction" or "ADR"). The NER is catalyzed by a
Nucleoside Monophosphate Kinase (or NMPK) that acts to transfer a high-energy phosphate from a substrate ribonucleotide triphosphate (NTP) or, in some cases, a deoxyribonucleotide triphosphate (dNTP), to adenosine diphosphate (ADP), thereby generating the ATP that is detected by the coupled ATP detection reaction or ADR. By the selective inclusion of a specific set of substrates, as described below, the NER may be configured to generate ATP in an NTP- (or dNTP-), NDP- (or dNDP-) or NMP- (or dNMP-) dependent fashion. In other words, by altering which substrates are present, and which particular NMPK enzyme is used to catalyze the NER, assays according to the present invention can be configured to qualitatively or quantitatively assay for the presence of virtually any specific, biologically-relevant NTP, NDP, NMP, dNTP, dNDP, or dNMP, with the exception of ATP. The reason that the reactions of the present invention cannot be used for the qualitative or quantitative analysis of ATP, is that the NER ultimately generates ATP, which is then detected and quantified by a coupled ADR.
[0046] As should be apparent to the skilled artisan, the ATP generated by an NER can be detected by any suitable means or method of ATP detection that is specific for ATP, and that hydrolyzes or otherwise destroys or removes the ATP being detected. One such ATP detection method involves the ATP-dependent generation of light by a second reaction catalyzed by an enzyme known as a "luciferase." This method is specific for ATP because firefly luciferases specifically utilize ATP as a substrate in the reaction that generates light.
[0047] In addition to the qualitative and quantitative analysis of specific NTPs, NDPs, NMPs, dNTPs, dNDPs, or dNMPs, the present invention can also be used to quantify the amount of nucleotide hydrolysis (other than ATP hydrolysis) catalyzed by a particular enzyme, when the hydrolysis results in the production of a nucleoside diphosphate or monophosphate that can be used as a substrate by a particular NMPK. The method works as follows: Nucleotide hydrolysis by the enzyme in under study generates a nucleotide substrate for a particular NMPK that, in turn, catalyzes an NER that generates ATP from ADP in a substrate-dependent fashion. The ATP so-generated is then utilized by an ADR, such as a luciferase-catalyzed production of light, to quantify the amount of ATP produced by the NER. The ADR must be specific for ATP, and for luciferase-catalyzed ADRs, this specificity results from the substrate specificity of luciferases, which utilize ATP, and no other nucleoside triphosphates, as a substrate in the reactions they catalyze to generate light. Under such conditions the nucleotide triphosphates (other than ATP) hydrolyzed by the enzyme under study do not contribute to the luminescent signal generated by luciferase, and thus, do not interfere with the "readout" portion of the assay.
[0048] In defining the disclosed assays and all related assays that can be developed using the disclosed inventive approach, the present invention provides reaction mixtures that may be used to conduct these assays. The present invention also provides kits that may be used for conducting such assays.
Nucleoside Monophosphate Kinases
[0049] Nucleoside monophosphate kinases (NMPKs) are a class of transferase enzymes, more specifically phosphotransferase enzymes, that catalyze the following reversible chemical reaction:
Figure imgf000012_0001
[0050] As depicted above, in one direction (the "forward reaction") this reaction involves the transfer of a high energy phosphate from ATP to a nucleoside
monophosphate (NMP) to form the corresponding nucleoside diphosphate (NDP) and ADP; while in the other direction (the "reverse reaction"), the reaction involves a transfer of a phosphate from NDP to ADP, thereby generating ATP and the corresponding NMP. It is the readily reversible nature of the reaction catalyzed by NMPKs that makes the approach and assays of the present invention possible.
Furthermore, it should be apparent that in either direction, the NMPK enzyme acts upon two substrates to form two products, and that the differences observed between the substrates and products involves the redistribution of a single phosphate group.
[0051] As noted, NMPKs fall into different subclasses that have evolved to catalyze the above reversible reaction using specific subsets of NMPs and NDPs. These subclasses of enzymes are named for the NMP/NDP substrates to which they add and remove phosphates by the phosphotransfer reactions they catalyze. For example, NMPKs that transfer phosphates from ATP to AMP in the forward reaction depicted above are known as adenosine monophosphate kinases, AMPKs, or adenylate kinases (AKs). NMPKs that transfer phosphates from ATP to GMP in the forward reaction depicted above are known as guanosine monophosphate kinases, GMPKs, or guanylate kinases (GKs). NMPKs that transfer phosphates from ATP to CMP in the forward reaction depicted above are known as cytidine monophosphate kinases, AMPKs, or cytidylate kinases (CKs). NMPKs that transfer phosphates from ATP to TMP in the forward reaction depicted above are known as thymidine monophosphate kinases, TMPKs, or thymidylate kinases (TKs). NMPKs that transfer phosphates from ATP to UMP in the forward reaction depicted above are known as uridine monophosphate kinases, UMPKs, or uridylate kinases (UKs). All of five of these subclasses of NMPKs have been classified by the International Union of Biochemistry and Molecular Biology (IUBMB) as members of enzyme class EC 2.7.4.4 and members of each subclass are found in all living organisms.
[0052] Biochemical studies have revealed that NMPKs generally follow a random Bi-Bi kinetic mechanism in catalyzing the above reaction in both directions. Structural studies have revealed that each NMPK molecule has two separate binding sites for the two substrates; one for the phosphate donor nucleotide (the NTP site), and the phosphate acceptor nucleotide (the NMP site). For a review, see Yan and Tsai;
Nucleoside Monphosphate Kinases: Structure, Mechanism, and Substrate Specificity; Adv. Enzymol. Relat. Areas Mol. Biol.; 73 : 103-134; 1999. [0053] In general, NMPKs are not very specific with respect to the phosphate donors they utilize. While most NMPKs prefer ATP at their NTP site (in some cases by an order of magnitude more than other NTPs), most NMPKs will utilize other NTPs, and even sometimes dNTPs, as phosphate donors for the forward reaction depicted above. X-Ray crystallographic studies of NMPK structures confirm that the low specificity of NMPKs for ATP at the NTP site is not unexpected since most of the observed binding energy come from the triphosphate moiety (Miiller et al., Structure 4: 147- 156, 1996) and the adenosine moiety of ATP is only loosely bound in all known crystal structures (Yan & Tsi; Adv. Enzymol. Relat. Areas Mol. Biol.; 73 : 103- 134;
1999). In fact, in these crystal structures, there is only one hydrogen bond observed between the adenosine moiety and the enzymes, which is likely responsible for the slight preference of ATP over other NTPs, and, in some cases, dNTPs. See: Yan & Tsi; Adv. Enzymol. Relat. Areas Mol. Biol.; 73 : 103-134; 1999.
[0054] In contrast to the NTP site, the NMP site of NMPKs has been found to make extensive interactions with the substrate NMP. In other words, while many NMPKs will utilize a variety of phosphate donors other than ATP, they tend to be quite specific for a given subclass of phosphate acceptors. Hence, for instance, AMPKs greatly prefer AMP as the phosphate acceptor in the forward reaction. However, there are notable exceptions to this preference. For example, while GMPKs greatly prefer GMP at their NMP binding site, some members of the family can utilize IMP as a phosphate acceptor. UMPKs appear to have the lowest specificity with respect to phosphate acceptors. They will readily catalyze phosphoryl transfer to CMP and AMP, in addition to UMP.
[0055] While most NMPKs show a high degree of specificity for their cognate phosphate acceptors at their NMP site, several NMPKs have been found by the inventors to tolerate deoxyribose-containing NMPs at this site. This means that in certain situations, dNMPs can be used as phosphate acceptors in the forward reaction, while dNDPs can be used as a source of phosphate to be transferred to ADP (forming ATP) in the reverse reaction.
[0056] Important to the adaptability of the design of the assays disclosed herein, the inventors have found that CMPK from Escherishia coli (E. coli) will readily use GTP, instead of CTP as a phosphate donor. Furthermore, the inventors have found that AMPK, GMPK and UMPK, also from E. coli, will effectively and readily utilize dATP as a phosphate donor, instead of their cognate respective phosphate donors, ATP, GTP and UTP. This "relaxed specificity" of NMPKs for specific phosphate donors at their NTP site greatly aids in the design of assays for nucleotides other than ATP. Similarly, the ability of NMPKs from E. coli to tolerate deoxyribose-containing NMPs at their NMP sites, means that these assays can be adapted for the detection and quantification of a wide variety of dNTPs, dNDPs and dNMPs, in addition to NTPs (other than ATP), NDPs, and NMPs. The importance of this fact will be further revealed in the Examples described below.
The "Nucleotide Exchange Reaction"
[0057] Importantly, the NMPK-catalyzed reaction depicted above is readily reversible. This makes it possible, under the appropriate conditions, to create a cyclic reaction, which is generally referred to herein as a Nucleotide Exchange Reaction, or NER (Figure 1). As depicted in Figure 1 , in the "forward" part of the NER {i. e. , top half), an NTP other than ATP, or if possible, a dNTP, is used as the phosphate donor {i.e. , "BTP") and a particular NMP, or if possible, dNMP, is used as the phosphate acceptor, resulting in the formation of the dephosphorylated BTP {i. e. , BDP) and the diphosphorylated NDP or dNDP. In the "reverse" part of the NER {i. e. , bottom half), the phosphate on the diphosphorylated NDP or dNDP is transferred to ADP, thereby creating a molecule of ATP and "regenerating" the starting phosphate acceptor NMP or dNMP.
[0058] To catalyze both the forward and reverse reactions of an NER as depicted Figure 1 , the NMPK used must possess the appropriate substrate specificity required. Since, as discussed above, most NMPKs will utilize a variety of phosphate donors but generally tend to be quite specific for a given subclass of phosphate acceptors, the type of NMPK chosen to catalyze the NER will largely be determined by the subclass of phosphate acceptors to be detected or quantified. Hence, should one wish to assay for GMP, GDP or GTP, GMPK would generally be chosen as the NMPK to catalyze the NER. In other words, the choice of subclass of NMPK to use for catalyzing the NER should generally match the subclass of phosphate acceptors used in the forward reaction (i.e. , GMPK for GMP, CMPK for CMP, etc.).
[0059] Conversely, due to the relative lack of specificity of NMPKs for the phosphate donors they bind in their NTP site, non-cognate BTPs can often be employed as the phosphate donor to "drive" the forward reaction by mass action. This fact, while subtle, is important, since it allows the forward reaction to be "driven" by a phosphate donor that, once converted to a diphosphate, will not be a favored substrate for the reverse reaction. The examples below provide several embodiments of the assays of the present invention where a non-cognate phosphate donor is used to drive the forward reaction of the NER.
[0060] Finally, it should be noted that it is the ATP generated by the reverse reaction (bottom half) of the NER that is used as an intermediary to couple the NER to some form of an ATP detection method or assay (i. e. , the ADR).
The Coupled ATP Detection Reaction
[0061] Figure 2 depicts how the ATP produced from one cycle of the NER can be used as an intermediary to couple the NER to a particular ATP detection reaction, or ADR. As depicted in Figure 2 the phosphate originally on the phosphate donor BTP is ultimately transferred to ADP to create the ATP that is detected by the "coupled" ADR.
[0062] In the embodiment depicted the ADR employed releases the chemical energy stored in ATP to generate light, as shown. The ATP produced by the NER is utilized by the enzyme luciferase, in the presence of the substrate luciferin and molecular oxygen (02) to generate light, AMP, PPi, Oxyluciferin and C02. The light produced by this reaction may be conveniently detected by any appropriate means, including photomultiplier tubes and other types of optical sensors, or by the exposure of photographic film. Since firefly luciferase only uses ATP as a substrate, and not other NTPs or dNTPs, other types of nucleoside triphosphates will not interfere with the ADR catalyzed by luciferase.
[0063] Importantly, the ADR consumes the ATP generated by the NER, in this case converting it to AMP and PPi. The AMP created by this ADR cannot be used as a substrate by the NMPK catalyzing the NER if the NMPK is not AMPK. If however, the NMPK being used is AMPK, the rate of the NER will still be limited by the quantity of BTP present.
[0064] As should be apparent to the skilled artisan, other means of detecting the ATP produced by the NER may be substituted for the ADR depicted in Figure 2.
Regardless of what type of ATP detection means is employed, the process involved must either consume the ATP created by the NER, or otherwise remove it from the reaction mixture, so as not to inhibit the NER by "product inhibition" that would result from increased concentrations of ATP.
Detection and Quantification of NMPs or dNMPs
[0065] Figure 3 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NMP or dNMP in a sample.
[0066] As depicted, in order for the NER to be dependent upon the NMP or dNMP to be assayed, and therefore indicative of the amounts of the NMP or dNMP to be quantified, a reaction mix must be assembled comprising:
(1) a cognate NMPK (i.e. , an NMPK having a phosphate acceptor specificity
matching that of the NMP or dNMP to be detected),
(2) a BTP that can either be from the same class of nucleotide as the NMP or dNMP to be detected, or preferably is from a different class of nucleotides, and
(3) ADP.
[0067] No other components of the NER need be present (as indicated by the lighter shading), since they will be generated by the NMPK during the course of the NER. However, the appropriate reagents of the chosen ADR should be included. In the present case, the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (02).
[0068] If configured as shown, the coupled NER and ADR will be dependent upon the NMP or dNMP to be assayed. Such NMP or dNMP, when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed forward reaction to proceed, thereby generating BDP and NDP or dNDP. The NDP or dNDP, so-generated, will allow the NMPK-catalyzed reverse reaction to proceed, and a phosphate will be transferred from the NDP or dNDP, to the ADP, thereby generating ATP. The ATP, so-generated, will be utilized by the luciferase, along with the luciferin and 02, to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C02, and light. The amount of light produced will ultimately be dependent upon the amount of NMP or dNMP added to the reaction mixture in the form of the added sample.
[0069] Importantly, if the sample to be added is not a purified sample free of "intrinsic ATP," but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted" as background from the amount detected by the coupled NER and ADR.
[0070] Examples 6, 7, and 8 (below) provide working examples of assays of the type described above, in which the presence and relative amounts of CMP, AMP, and UMP are, respectively, detected.
Detection and Quantification of NDPs or dNDPs
[0071] Figure 4 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NDP or dNDP in a sample. Note that in this depiction, as in this configuration, the NMPK is used exclusively to catalyze the reverse reaction; however, BTP can be included to facilitate amplification of the signal generated.
[0072] As depicted, in order for the NER to be dependent upon the NDP or dNDP to be assayed, and therefore indicative of the amounts of the NDP or dNDP to be quantified, a reaction mix must be assembled comprising:
(1) a cognate NMPK (i.e. , an NMPK having a phosphate acceptor specificity
matching that of the NDP or dNDP to be detected), and
(2) ADP.
[0073] As noted above, a BTP can be included, but is optional. If included, the BTP can either be from the same class of nucleotide as the NDP or dNDP to be detected, or preferably is from a different class of nucleotides. Inclusion of a BTP can serve to amplify the signal, since the NMP or dNMP generated by the reverse reaction will serve as a phosphate acceptor in a subsequent forward reaction wherein the phosphate is donated by BTP.
[0074] No other components of the NER need be present (as indicated by the lighter shading), since they will be generated by the NMPK during the course of the NER. However, the appropriate reagents of the chosen ADR should be included. In the present case, the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (02).
[0075] If configured as shown, the coupled NER and ADR will be dependent upon the NDP or dNDP to be assayed. Such NDP or dNDP, when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed reverse reaction to proceed, thereby generating ATP, and NMP or dNMP. The ATP, so- generated, will be utilized by the luciferase, along with the luciferin and 02, to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C02, and light. The amount of light produced will ultimately be dependent upon the amount of NDP or dNDP added to the reaction mixture in the form of the added sample.
[0076] As noted above, if the sample to be added is not a purified sample free of "intrinsic ATP," but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted" as background from the amount detected by the coupled NER and ADR.
[0077] Examples 3 , 7, and 8 (below) provide working examples of assays of the type described above, in which the presence and relative amounts of GDP, ADP, and UDP are, respectively, detected.
Detection and Quantification of NTPs Other Than ATP (i.e., BTPs)
[0078] Figure 5 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular NTP other than ATP (i. e. , BDP) in a sample. [0079] As depicted, in order for the NER to be dependent upon the BTP to be assayed, and therefore indicative of the amounts of the BTP to be quantified, a reaction mix must be assembled comprising:
(1) an NMPK that can effectively utilize the BTP to be detected as a phosphate
donor, and that has a phosphate acceptor specificity that matches the NMP or dNMP to be included in the reaction,
(2) cognate NMP or dNMP (i. e. , matching the phosphate acceptor specificity of the NMPK to be used), and
(3) ADP.
[0080] No other components of the NER need be present (as indicated by the lighter shading), since they will be generated by the NMPK during the course of the NER. However, the appropriate reagents of the chosen ADR should be included. In the present case, the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (02).
[0081] If configured as shown, the coupled NER and ADR will be dependent upon the BTP to be assayed. Such BTP, when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed forward reaction to proceed, and will provide the phosphate to be transferred to the included NMP or dNMP. The forward reaction will thereby generate BDP and NDP or dNDP. The NDP or dNDP, so- generated, will allow the NMPK-catalyzed reverse reaction to proceed, and a phosphate will be transferred from the NDP or dNDP, to the ADP, thereby generating ATP. The ATP, so-generated, will be utilized by the luciferase, along with the luciferin and 02, to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C02, and light. The amount of light produced will ultimately be dependent upon the amount of BTP added to the reaction mixture in the form of the added sample.
[0082] As noted above, if the sample to be added is not a purified sample free of "intrinsic ATP," but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted" as background from the amount detected by the coupled NER and ADR. [0083] Example 2 (below) provides a working example of assays of the type described above, in which the presence and relative amounts of GTP (and GDP) are detected.
Detection and Quantification of Cyclic Nucleotides
[0084] Figure 6 depicts how the coupled NER and ADR of the invention may be configured to specifically detect and quantify the amount of a particular cyclic nucleotide in a sample. In this case the cyclic nucleotide (i. e. , cNMP) must first be converted to a standard 5 '-phosporylated NMP. This conversion reaction is
accomplished by the inclusion of a cyclic nucleotide phosphodiesterase with
appropriate substrate specificity.
[0085] Following conversion of the cNMP to NMP, the resulting NMP is detected using the same approach as depicted in Figure 3, and outlined above in the section entitled Detection and Quantification of NMPs or dNMPs.
[0086] As noted above, if the sample to be added is not a purified sample free of "intrinsic ATP," but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted" as background from the amount detected by the coupled NER and ADR.
Quantification of NTPase Activity
[0087] Figures 7 and 8 depict two variations on how the coupled NER and ADR of the invention may be configured to specifically quantify the amount of a NTP hydrolysis catalyzed by an NTPase present in a sample, for example, a GTPase. In both cases, the GTPase generates GDP, which is then used to drive the reverse reaction catalyzed by GMPK. In effect the GDP generated by the GTPase catalyzed hydrolysis is detected using the same approach depicted in Figure 4, and outlined in the section entitled Detection and Quantification of NDPs or dNDPs.
[0088] In Figure 7, a generalized scheme is depicted in which a BTP other than ATP, and other than GTP is provided to amplify the signal generated. Importantly, most GTPases have a significantly higher affinity for GTP than does GMPK, which normally would utilize ATP as a phosphate donor, and GMP as a phosphate acceptor in the forward reaction. Inclusion of excess BTP, however, would effectively compete with GTP for use as a phosphate donor. In Figure 8, the specific BTP used is dATP, which can be used as a phosphate donor by GMPK, but is not used as a substrate by luciferase.
[0089] In order for the NER depicted in Figures 7 and 8 to be dependent upon the GDP generated by the GTPase, and therefore indicative of the amount of GTPase activity present, a reaction mix must be assembled comprising:
(1) GMPK, and
(2) ADP.
[0090] As show in Figure 7, a BTP (such as dATP, as shown in Figure 8) can be included, but is optional. If included, the BTP is preferably from a different class of nucleotides than GTP (such as dATP), and is included in excess over GTP. However, as noted above, in most cases, the GTPase being assayed would have a significantly greater affinity for GTP than the GMPK. Also as noted, inclusion of a BTP serves to amplify the signal, since the GMP generated by the reverse reaction will serve as a phosphate acceptor in a subsequent forward reaction wherein the phosphate is donated by BTP.
[0091] No other components of the NER need be present (as indicated by the lighter shading), since they will be generated by the NMPK during the course of the GTPase reaction and NER. However, the appropriate reagents of the chosen ADR should be included. In the present case, the reagents of the ADR that should be present in the reaction mixture are the luciferase enzyme, luciferin, and sufficient molecular oxygen (02).
[0092] If configured as shown, the coupled NER and ADR will be dependent upon the GDP produced by the hydrolysis of GTP catalyzed by the GTPase. Such GDP, when added to the reaction mixture in the form of an added sample, will allow the NMPK-catalyzed reverse reaction to proceed, thereby generating ATP, and GMP. The ATP, so-generated, will be utilized by the luciferase, along with the luciferin and 02, to drive the ADR, thereby generating AMP, PPi, Oxyluciferin, C02, and light. The amount of light produced will ultimately be dependent upon the amount of GDP produced by the GTPase in the added sample.
[0093] As noted above, if the sample to be added is not a purified sample free of "intrinsic ATP," but rather is a biological sample, the sample must either first be depleted of any intrinsic ATP that would interfere with the ADR read-out, or else the level of intrinsic ATP must be determined using the ADR of choice (without the coupled NER), so that the amount of intrinsic ATP present can be "subtracted" as background from the amount detected by the coupled NER and ADR.
[0094] Example 4 (below) provides a working example of assays of the type described above, although no additional BTP or dATP was added, demonstrating the optional nature of the BTP or dATP addition.
Detection of Inhibitors of NTPase Activity
[0095] As mentioned above, the methods and assays present invention can be adapted for use in screening assays, such as assays designed to detect the inhibition of particular NTPases, including GTPases. Such screening assays can be used to detect and study inhibitors of specific NTPases.
[0096] By way of example, the present invention provides methods of screening test compounds for their ability to inhibit a particular GTPase present in a sample.
Such methods comprising combining said sample with a test compound, GTP, a purified guanosine monophosphate kinase (GMPK), ADP; and optionally, an NTP, other than ATP or GTP, capable of use as a phosphate donor by the GMPK, and detecting any ATP generated. The amount of ATP generated is indicative of the amount or activity of the particular GTPase present in said sample, and a reduction of the ATP generated in the presence of said test compound, relative to the amount generated in the absence of said test compound, indicates that said test compound inhibits said particular GTPase.
[0097] Using similar methods it is conceivable to screen for inhibitors of virtually any enzyme that consumes or generates nucleotides other than ATP. Such screening assays can be configured according to thenucleotide substrate requirements of the enzyme being studied, and a cognate NMPK is employed to catalyze the relevant NER. Enzymes Whose Activities May be Assayed Using the Methods of the Invention
[0098] The following is a list of 324 classes of enzymes - listed by the reactions they catalyze and identified by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMC) Enzyme Code (EC) and accepted name - whose activities may conceivably be assayed using the methods of the present invention, as modified according to one or more of the several permutations described herein. Particularly, the activities of the enzymes in these classes may be assayed by monitoring the amount of nucleotides specifically produced or consumed during the enzymatic reactions catalyzed by these enzymes.
[0099] It should be noted that while this is a substantial list of enzyme classes, it is not meant to be taken as an exhaustive or exclusive list of specific enzymes. It should also be noted that the methods of the present invention have the potential to be used to assay the activities of other nucleotide-consuming or nucleotide-generating enzymes not specifically encompassed by the classes of enzymes listed below.
No. IUBMC Enzyme Code & Name
1 EC 1.7. 1.7 GMP reductase
2 EC 2.4. 1.1 1 glycogen(starch) synthase
3 EC 2.4. 1.12 cellulose synthase (UDP-forming)
4 EC 2.4. 1 .13 sucrose synthase
5 EC 2.4. 1 .14 sucrose-phosphate synthase
6 EC 2.4. 1.15 α,α-trehalose-phosphate synthase (UDP-forming)
7 EC 2.4. 1.16 chitin synthase
8 EC 2.4. 1.17 glucuronosyltransferase
9 EC 2.4. 1.21 starch synthase
10 EC 2.4. 1.22 lactose synthase
1 1 EC 2.4. 1.23 sphingosine β-galactosyltransferase
12 EC 2.4. 1.26 DNA a-glucosyltransferase
13 EC 2.4. 1.27 DNA β-glucosyltransferase
14 EC 2.4. 1 .28 glucosyl-DNA β-glucosyltransferase IUBMC Enzyme Code & Name
EC 2.4. 1.29 cellulose synthase (GDP-forming)
EC 2.4. 1.32 glucomannan 4-P-mannosyltransferase
EC 2.4. 1.33 alginate synthase
EC 2.4. 1.34 1 ,3-P"glucan synthase
EC 2.4. 1 .35 phenol β-glucosyltransferase
EC 2.4. 1.36 α,α-trehalose-phosphate synthase (GDP-forming)
EC 2.4. 1 .37 fucosylgalactoside 3-a-galactosyltransferase
EC 2.4. 1.38 β-Ν-acetylglucosaminyl-glycopeptide β- 1 ,4-galactosyltransferase
EC 2.4. 1.39 steroid N-acetylglucosaminyltransferase
EC 2.4. 1.40 glycoprotein-fucosylgalactoside a-N- acetylgalactosaminyltransferase
EC 2.4. 1.41 polypeptide N-acetylgalactosaminyltransferase
EC 2.4. 1.43 polygalacturonate 4-a-galacturonosyltransferase
EC 2.4. 1.44 lipopolysaccharide 3-a-galactosyltransferase
EC 2.4. 1.45 2-hydroxyacylsphingosine Ι -β-galactosyltransferase
EC 2.4. 1.46 monogalactosyldiacylglycerol synthase
EC 2.4. 1.47 N-acylsphingosine galactosyltransferase
EC 2.4. 1.48 heteroglycan a-mannosyltransferase
EC 2.4. 1 .52 poly(glycerol-phosphate) a-glucosyltransferase
EC 2.4. 1.53 poly(ribitol-phosphate) β-glucosyltransferase
EC 2.4. 1.54 undecaprenyl-phosphate mannosyltransferase
EC 2.4. 1.56 lipopolysaccharide N-acetylglucosaminyltransferase
EC 2.4. 1.57 phosphatidylinositol a-mannosyltransferase
EC 2.4. 1.58 lipopolysaccharide glucosyltransferase I
EC 2.4. 1.60 abequosyltransferase
EC 2.4. 1.62 ganglioside galactosyltransferase
EC 2.4. 1.63 linamarin synthase
EC 2.4. 1.65 3-galactosyl-N-acetylglucosaminide 4-a-L-fucosyltransferase
EC 2.4. 1 .66 procollagen glucosyltransferase
EC 2.4. 1 .68 glycoprotein 6-a-L-fucosyltransferase IUBMC Enzyme Code & Name
EC 2 .4. 1.69 galactoside 2-a-L-fucosyltransferase
EC 2 .4. 1.70 poly(ribitol-phosphate) N-acetylglucosaminyltransferase
EC 2 .4. 1.71 arylamine glucosyltransferase
EC 2 .4. 1.73 lipopolysaccharide glucosyltransferase II
EC 2 .4. 1 .74 glycosaminoglycan galactosyltransferase
EC 2 .4. 1.78 phosphopolyprenol glucosyltransferase
EC 2 .4. 1.79 globotriaosylceramide 3-P-N-acetylgalactosaminyltransferase
EC 2 .4. 1.80 ceramide glucosyltransferase
EC 2 .4. 1.81 flavone 7-0-P-glucosyltransferase
EC 2 .4. 1.83 dolichyl-phosphate β-D-mannosyltransferase
EC 2 .4. 1.85 cyanohydrin β-glucosyltransferase
EC 2 .4. 1 .86 glucosaminylgalactosylglucosylceramide β-galactosyltransferase
EC 2 .4. 1.87 N-acetyllactosaminide 3-a-galactosyltransferase
EC 2 .4. 1.88 globoside a-N-acetylgalactosaminyltransferase
EC 2 .4. 1.90 N-acetyllactosamine synthase
EC 2 .4. 1.91 flavonol 3-O-glucosyltransferase
EC 2 .4. 1.92 (N-acetylneuraminyl)-galactosylglucosylceramide N- acetylgalactosaminyltransferase
EC 2.4.1.94 protein N-acetylglucosaminyltransferase
EC 2.4.1.96 sn-glycerol-3-phosphate 1 -galactosyltransferase
EC 2.4.1.101 a- l ,3-mannosyl-glycoprotein 2-β-Ν- acetylglucosaminyltransferase
EC 2.4.1.102 β-l ,3-galactosyl-0-glycosyl-glycoprotein β-1 ,6-Ν- acetylglucosaminyltransferase
EC 2.4.1.103 alizarin 2^-glucosyltransferase
EC 2.4.1.104 o-dihydroxycoumarin 7-O-glucosyltransferase
EC 2.4.1.105 vitexin β-glucosyltransferase
EC 2.4.1.106 isovitexin β-glucosyltransferase
EC 2.4.1.1 10 tRNA-queuosine β-mannosyltransferase
EC 2.4.1.1 1 1 coniferyl-alcohol glucosyltransferase IUBMC Enzyme Code & Name
71 EC 2 .4. 1 .1 12 a- l ,4-glucan-protein synthase (UDP-forming)
72 EC 2 .4. 1 .1 13 a- l ,4-glucan-protein synthase (ADP-forming)
73 EC 2 .4. 1 .1 14 2-coumarate Ο-β-glucosyltransferase
74 EC 2 .4. 1 .1 15 anthocyanidin 3-O-glucosyltransferase
75 EC 2 .4. 1 .1 16 cyanidin 3-O-rutinoside 5-O-glucosyltransferase
76 EC 2 .4. 1 .1 17 dolichyl-phosphate β-glucosyltransferase
77 EC 2 .4. 1 , .1 18 cytokinin 7-P-glucosyltransferase
78 EC 2 .4. 1 .120 sinapate 1 -glucosyltransferase
79 EC 2 .4. 1 .121 indole-3-acetate β-glucosyltransferase
80 EC 2 .4. 1 , .122 glycoprotein-N-acetylgalactosamine 3-P-galactosyltransferase
81 EC 2 .4. 1 .123 inositol 3-a-galactosyltransferase
82 EC 2 .4. 1 .126 hydroxycinnamate 4-P-glucosyltransferase
83 EC 2 .4. 1 .127 monoterpenol β-glucosyltransferase
84 EC 2 .4. 1 , .128 scopoletin glucosyltransferase
85 EC 2 .4. 1 .131 glycolipid 2-a-mannosyltransferase
86 EC 2 .4. 1 .132 glycolipid 3-a-mannosyltransferase
87 EC 2 .4. 1 .133 xylosylprotein 4-P-galactosyltransferase
88 EC 2 .4. 1 .134 galactosylxylosylprotein 3-P-galactosyltransferase
89 EC 2 .4. 1 .135 galactosylgalactosylxylosylprotein 3-P-glucuronosyltransferase
90 EC 2 .4. 1 .136 gallate Ι -β-glucosyltransferase
91 EC 2 .4. 1 .137 sn-glycerol-3-phosphate 2-a-galactosyltransferase
92 EC 2 .4. 1 , .138 mannotetraose 2-a-N-acetylglucosaminyltransferase
93 EC 2 .4. 1 .141 N-acetylglucosaminyldiphosphodolichol N- acetylglucosaminyltransferase
94 EC 2.4.1.142 chitobiosyldiphosphodolichol β-mannosyltransferase
chitobiosyldiphosphodolichol a-mannosyltransferase
95 EC 2.4.1.143 a- l ,6-mannosyl-glycoprotein 2-β-Ν- acetylglucosaminyltransferase
96 EC 2.4.1.144 P-l ,4-mannosyl-glycoprotein 4-β-Ν- acetylglucosaminyltransferase No. IUBMC Enzyme Code & Name
97 EC 2.4.1.145 a- l ,3-mannosyl-glycoprotein 4-β-Ν- acetylglucosaminyltransferase
98 EC 2.4.1.146 P-l ,3-galactosyl-0-glycosyl-glycoprotein β-1 ,3-Ν- acetylglucosaminyltransferase
99 EC 2.4.1.147 acetylgalactosaminyl-O-glycosyl-glycoprotein β- 1 ,3-Ν- acetylglucosaminyltransferase
100 EC 2.4.1.148 acetylgalactosaminyl-O-glycosyl-glycoprotein β- 1 ,6-Ν- acetylglucosaminyltransferase
101 EC 2.4.1.149 N-acetyllactosaminide β-l ,3-N-acetylglucosaminyltransferase
102 EC 2.4.1.150 N-acetyllactosaminide β-l ,6-N-acetylglucosaminyl-transferase
103 EC 2.4.1.152 galactoside 3-fucosyltransferase
104 EC 2.4.1.153 dolichyl-phosphate a-N-acetylglucosaminyltransferase
105 EC 2.4.1.155 a- l ,6-mannosyl-glycoprotein 6-β-Ν- acetylglucosaminyltransferase
106 EC 2.4.1.156 indolylacetyl-myo-inositol galactosyltransferase
107 EC 2.4.1.157 1 ,2-diacylglycerol 3-glucosyltransferase
108 EC 2.4.1.158 13 -hydroxy docosanoate 13^-glucosyltransferase
109 EC 2.4.1.159 f avonol-3-O-glucoside L-rhamnosyltransferase
1 10 EC 2.4.1.160 pyridoxine 5'-0^-D-glucosyltransferase
1 1 1 EC 2.4.1.163 β-galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide β- 1 ,3-acetylglucosaminyltransferase
1 12 EC 2.4.1.164 galactosyl-N-acetylglucosaminylgalactosylglucosyl-ceramide β- 1 ,6-N-acetylglucosaminyltransferase
1 13 EC 2.4.1.165 N-acetylneuraminylgalactosylglucosylceramide β- 1 ,4-Ν- acetylgalactosaminyltransferase
1 14 EC 2.4.1.167 sucrose 6F-a-galactosyltransferase
1 15 EC 2.4.1.168 xyloglucan 4-glucosyltransferase
1 16 EC 2.4.1.170 isof avone 7-O-glucosyltransferase
1 17 EC 2.4.1.171 methyl-ONN-azoxymethanol β-D-glucosyltransferase
1 18 EC 2.4.1.172 salicyl-alcohol β-D-glucosyltransferase IUBMC Enzyme Code & Name
EC 2.4.1.173 sterol 3 P-glucosyltransferase
EC 2.4.1 .174 glucuronylgalactosylproteoglycan 4-β-Ν- acetylgalactosaminyltransferase
EC 2.4.1.175 glucuronosyl-N-acetylgalactosaminyl-proteoglycan 4-β-Ν- acetylgalactosaminyltransferase
EC 2.4.1.176 gibberellin β-D-glucosyltransferase
EC 2.4.1.177 cinnamate β-D-glucosyltransferase
EC 2.4.1.178 hydroxymandelonitrile glucosyltransferase
EC 2.4.1.179 lactosylceramide β-l ,3-galactosyltransferase
EC 2.4.1.180 lipopolysaccharide N-acetylmannosaminouronosyltransferase EC 2.4.1.181 hydroxyanthraquinone glucosyltransferase
EC 2.4.1.182 lipid- A-disaccharide synthase
EC 2.4.1.183 a- l ,3-glucan synthase
EC 2.4.1.185 flavanone 7-0^-glucosyltransferase
EC 2.4.1.186 glycogenin glucosyltransferase
EC 2.4.1.187 N-acetylglucosaminyldiphosphoundecaprenol N-acetyl-β-ϋ- mannosaminyltransferase
EC 2.4.1.188 N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase EC 2.4.1.189 luteolin 7-O-glucuronosyltransferase
EC 2.4.1.190 luteolin-7-O-glucuronide 2"-0-glucuronosyltransferase
EC 2.4.1.191 luteolin-7-O-diglucuronide 4'-0-glucuronosyltransferase
EC 2.4.1.192 nuatigenin 3 β-glucosyltransferase
EC 2.4.1.193 sarsapogenin 3β-glucosyltransferase
EC 2.4.1.194 4-hydroxybenzoate 4-0^-D-glucosyltransferase
EC 2.4.1 .195 N-hydroxythioamide S^-glucosyltransferase
EC 2.4.1.196 nicotinate glucosyltransferase
EC 2.4.1.197 high-mannose-oligosaccharide β- 1 ,4-Ν- acetylglucosaminyltransferase
EC 2.4.1.198 phosphatidylinositol N-acetylglucosaminyltransferase
EC 2.4.1.201 a- l ,6-mannosyl-glycoprotein 4-β-Ν- o. IUBMC Enzyme Code & Name
acetylglucosaminyltransferase
145 EC 2.4.1.202 2,4-dihydroxy-7-methoxy-2H- l ,4-benzoxazin-3(4H)-one 2 -D- glucosyltransferase
146 EC 2.4.1.203 trans-zeatin Ο-β-D-glucosyltransferase
147 EC 2.4.1.205 galactogen 6P-galactosyltransferase
148 EC 2.4.1.206 lactosylceramide l ,3-N-acetyl-P-D-glucosaminyltransferase
149 EC 2.4.1 .208 diglucosyl diacylglycerol synthase
150 EC 2.4.1.209 cis-p-coumarate glucosyltransferase
151 EC 2.4.1.210 limonoid glucosyltransferase
152 EC 2.4.1.212 hyaluronan synthase
153 EC 2.4.1 .213 glucosylglycerol-phosphate synthase
154 EC 2.4.1 .214 glycoprotein 3-a-L-fucosyltransferase
155 EC 2.4.1.215 cis-zeatin Ο-β-D-glucosyltransferase
156 EC 2.4.1.217 mannosyl-3-phosphoglycerate synthase
157 EC 2.4.1.218 hydroquinone glucosyltransferase
158 EC 2.4.1.219 vomilenine glucosyltransferase
159 EC 2.4.1.220 indoxyl-UDPG glucosyltransferase
160 EC 2.4.1.221 peptide-O-fucosyltransferase
161 EC 2.4.1.222 O-fucosylpeptide 3-P-N-acetylglucosaminyltransferase
162 EC 2.4.1.223 glucuronyl-galactosyl-proteoglycan 4-a-N- acetylglucosaminyltransferase
163 EC 2.4.1.224 glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-a-N- acetylglucosaminyltransferase
164 EC 2.4.1.225 N-acetylglucosaminyl-proteoglycan 4-P-glucuronosyltransferase
165 EC 2.4.1.226 N-acetylgalactosaminyl-proteoglycan 3-P-glucuronosyltransferase
166 EC 2.4.1.227 undecaprenyldiphospho-muramoylpentapeptide β-Ν- acetylglucosaminyltransferase
167 EC 2.4.1.228 lactosylceramide 4-a-galactosyltransferase
168 EC 2.4.1.229 [Skp l -protein]-hydroxyproline N-acetylglucosaminyltransferase
169 EC 2.4.1.232 glycolipid 6-a-mannosyltransferase o. IUBMC Enzyme Code & Name
170 EC 2.4.1.234 kaempferol 3-O-galactosyltransferase
171 EC 2.4.1.236 flavanone 7-O-glucoside 2"-0-P-L-rhamnosyltransferase
172 EC 2.4.1.237 flavonol 7-0-P-glucosyltransferase
173 EC 2.4.1.238 anthocyanin 3'-0-P-glucosyltransferase
174 EC 2.4.1.239 flavonol-3-O-glucoside glucosyltransferase
175 EC 2.4.1.240 flavonol-3-O-glycoside glucosyltransferase
176 EC 2.4.1.241 digalactosyldiacylglycerol synthase
177 EC 2.4.1.242 NDP-glucose— starch glucosyltransferase
178 EC 2.4.1.244 N-acetyl-P-glucosaminyl-glycoprotein 4-β-Ν- acetylgalactosaminyltransferase
179 EC 2.4.1.245 α,α-trehalose synthase
180 EC 2.4.1.246 mannosylfructose-phosphate synthase
181 EC 2.4.2.7 adenine phosphoribosyltransferase
182 EC 2.4.2.9 uracil phosphoribosyltransferase
183 EC 2.4.2.24 1 ,4-P-D-xylan synthase
184 EC 2.4.2.25 flavone apiosyltransferase
185 EC 2.4.2.26 protein xylosyltransferase
186 EC 2.4.2.27 dTDP-dihydrostreptose— streptidine-6-phosphate
dihydrostreptosyltransferase
187 EC 2. .4, .2.32 dolichyl-phosphate D-xylosyltransferase
188 EC 2, .4, .2.34 indolylacetylinositol arabinosyltransferase
189 EC 2, .4, .2.35 flavonol-3-O-glycoside xylosyltransferase
190 EC 2, .4, .2.38 glycoprotein 2-P-D-xylosyltransferase
191 EC 2, .4, .2.39 xyloglucan 6-xylosyltransferase
192 EC 2. .4, .2.40 zeatin Ο-β-D-xylosyltransferase
193 EC 2, .4, .99.1 β-galactoside a-2,6-sialyltransferase
194 EC 2, .4, .99.2 monosialoganglioside sialyltransferase
195 EC 2, .4, .99.3 a-N-acetylgalactosaminide a-2,6-sialyltransferase
196 EC 2, .4, .99.4 β-galactoside a-2,3-sialyltransferase
197 EC 2. .4, .99.5 galactosyldiacylglycerol a-2,3-sialyltransferase No. IUBMC Enzyme Code & Name
198 EC 2.4.99.6 N-acetyllactosaminide a-2,3-sialyltransferase
199 EC 2.4.99.7 a-N-acetylneuraminyl-2,3-P-galactosyl- l ,3-N- acetylgalactosaminide 6-a-sialyltransferase
200 EC 2.4.99.8 a-N-acetylneuraminate a-2,8-sialyltransferase
201 EC 2.4.99.9 lactosylceramide a-2,3-sialyltransferase
202 EC 2.4.99.10 neolactotetraosylceramide a-2,3-sialyltransferase
203 EC 2.4.99.1 1 lactosylceramide a-2,6-N-sialyltransferase
204 EC 2.5.1.27 adenylate dimethylallyltransferase
205 EC 2.6.99.1 dATP(dGTP)— DNA purinetransferase
206 EC 2.7.1.81 hydroxylysine kinase
207 EC 2.7.1.108 dolichol kinase
208 EC 2.7.1.1 14 AMP— thymidine kinase
209 EC 2.7.1.1 18 ADP— thymidine kinase
210 EC 2.7.1.146 ADP-dependent phosphofructokinase
21 1 EC 2.7.1.156 adenosylcobinamide kinase
212 EC 2.7.1 .161 CTP-dependent riboflavin kinase
213 EC 2.7.2.10 phosphoglycerate kinase (GTP)
214 EC 2.7.7.9 UTP— glucose- 1 -phosphate uridylyltransferase
215 EC 2.7.7.10 UTP— hexose- 1 -phosphate uridylyltransferase
216 EC 2.7.7.1 1 UTP— xylose- 1 -phosphate uridylyltransferase
217 EC 2.7.7.13 mannose- 1 -phosphate guanylyltransferase
218 EC 2.7.7.14 ethanolamine-phosphate cytidylyltransferase
219 EC 2.7.7.15 choline-phosphate cytidylyltransferase
220 EC 2.7.7.21 tRNA cytidylyltransferase
221 EC 2.7.7.22 mannose- 1 -phosphate guanylyltransferase (GDP)
222 EC 2.7.7.23 UDP-N-acetylglucosamine diphosphorylase
223 EC 2.7.7.24 glucose- 1 -phosphate thymidylyltransferase
224 EC 2.7.7.28 nucleoside-triphosphate-hexose- 1 -phosphate nucleotidyltransferase
225 EC 2.7.7.30 fucose-1 -phosphate guanylyltransferase
226 EC 2.7.7.31 DNA nucleotidylexotransferase No. IUBMC Enzyme Code & Name
227 EC 2, .7, .7.32 galactose- 1 -phosphate thymidylyltransferase
228 EC 2, .7, .7.33 glucose- 1 -phosphate cytidylyltransferase
229 EC 2, .7, .7.34 glucose- 1 -phosphate guanylyltransferase
230 EC 2, .7, .7.37 aldose- 1 -phosphate nucleotidyltransferase
231 EC 2, .7, .7.38 3-deoxy-manno-octulosonate cytidylyltransferase
232 EC 2, .7, .7.39 glycerol-3-phosphate cytidylyltransferase
233 EC 2, .7, .7.40 D-ribitol-5-phosphate cytidylyltransferase
234 EC 2. .7, .7.41 phosphatidate cytidylyltransferase
235 EC 2, .7, .7.43 N-acylneuraminate cytidylyltransferase
236 EC 2, .7, .7.44 glucuronate- 1 -phosphate uridylyltransferase
237 EC 2. .7, .7.45 guanosine-triphosphate guanylyltransferase
238 EC 2. .7, .7.46 gentamicin 2"-nucleotidyltransferase
239 EC 2, .7, .7.48 RNA-directed RNA polymerase
240 EC 2, .7, .7.49 RNA-directed DNA polymerase
241 EC 2, .7, .7.50 mRNA guanylyltransferase
242 EC 2, .7, .7.62 adenosylcobinamide-phosphate guanylyltransferase
243 EC 2, .7, .7.65 diguanylate cyclase
244 EC 2, .7, .8.1 ethanolaminephosphotransferase
245 EC 2, .7, .8.2 diacylglycerol cholinephosphotransferase
246 EC 2, .7, .8.3 ceramide cholinephosphotransferase
247 EC 2, .7, .8.4 serine-phosphoethanolamine synthase
248 EC 2, .7, .8.5 CDP-diacylglycerol— glycerol-3-phosphate 3- phosphatidyltransferase
249 EC 2.7.8.6 undecaprenyl-phosphate galactose phosphotransferase
250 EC 2.7.8.8 CDP-diacylglycerol— serine O-phosphatidyltransferase
251 EC 2.7.8.9 phosphomannan mannosephosphotransferase
252 EC 2.7.8.10 sphingosine cholinephosphotransferase
253 EC 2.7.8.1 1 CDP-diacylglycerol— inositol 3-phosphatidyltransferase
254 EC 2.7.8.12 CDP-glycerol glycerophosphotransferase
255 EC 2.7.8.13 phospho-N-acetylmuramoyl-pentapeptide-transferase No. IUBMC Enzyme Code & Name
256 EC 2.7.8.14 CDP-ribitol ribitolphosphotransferase
257 EC 2.7.8.15 UDP-N-acetylglucosamine— dolichyl-phosphate N- acetylglucosaminephosphotransferase
258 EC 2.7.8.17 UDP-N-acetylglucosamine— lysosomal-enzyme N- acetylglucosaminephosphotransferase
259 EC 2.7.8.18 UDP-galactose— UDP-N-acetylglucosamine galactose phosphotransferase
260 EC 2.7.8.19 UDP-glucose— glycoprotein glucose phosphotransferase
261 EC 2.7.8.22 l -alkenyl-2-acylglycerol choline phosphotransferase
262 EC 2.7.8.24 phosphatidylcholine synthase
263 EC 2.7.8.26 adenosylcobinamide-GDP ribazoletransferase
264 EC 3.1.3.35 thymidylate 5'-phosphatase
265 EC 3.1.4.1 phosphodiesterase I
266 EC 3.1.4.16 2',3'-cyclic-nucleotide 2'-phosphodiesterase
267 EC 3.1.4.17 3',5'-cyclic-nucleotide phosphodiesterase
268 EC 3.1.4.35 3',5*-cyclic-GMP phosphodiesterase
269 EC 3.1.4.37 2',3'-cyclic-nucleotide 3'-phosphodiesterase
270 EC 3.1.4.40 CMP-N-acylneuraminate phosphodiesterase
271 EC 3.1.4.52 cyclic-guanylate-specific phosphodiesterase
272 EC 3.1 .4.53 3',5'-cyclic-AMP phosphodiesterase
273 EC 3.1.5.1 dGTPase
274 EC 3.1.13.4 poly(A)-specific ribonuclease
275 EC 3.1.13.5 ribonuclease D
276 EC 3. 1. 16.1 spleen exonuclease
277 EC 3.2.1 .42 GDP-glucosidase
278 EC 3.2.2.4 AMP nucleosidase
279 EC 3.5.4.6 AMP deaminase
280 EC 3.5.4.7 ADP deaminase
281 EC 3.5.4.12 dCMP deaminase
282 EC 3.5.4.13 dCTP deaminase No. IUBMC Enzyme Code & Name
283 EC 3. 5.4.16 GTP cyclohydrolase I
284 EC 3. 5.4.17 adenosine-phosphate deaminase
285 EC 3. .5.4.25 GTP cyclohydrolase II
286 EC 3. ,5.4.29 GTP cyclohydrolase Iia
287 EC 3. ,5.4.30 dCTP deaminase (dUMP-forming)
288 EC 3. .6.1 .12 dCTP diphosphatase
289 EC 3. ,6.1 .13 ADP-ribose diphosphatase
290 EC 3 .6.1.15 nucleoside-triphosphatase
291 EC 3 .6.1.16 CDP-glycerol diphosphatase
292 EC 3. ,6. 1.17 bis(5'-nucleosyl)-tetraphosphatase (asymmetrical)
293 EC 3 .6.1 .18 FAD diphosphatase
294 EC 3 .6.1.19 nucleoside-triphosphate diphosphatase
295 EC 3 .6.1.20 5'-acylphosphoadenosine hydrolase
296 EC 3 .6.1.21 ADP-sugar diphosphatase
297 EC 3 .6. 1.22 NAD diphosphatase
298 EC 3 .6.1 .23 dUTP diphosphatase
299 EC 3 .6.1.26 CDP-diacylglycerol-diphosphatase
300 EC 3 .6.1.29 bis(5'-adenosyl)-triphosphatase
301 EC 3 .6. 1.39 thymidine-triphosphatase
302 EC 3 .6.1 .40 guanosine-5'-triphosphate,3'-diphosphate diphosphatase
303 EC 3 .6.1.41 bis(5'-nucleosyl)-tetraphosphatase (symmetrical)
304 EC 3 .6.1 .42 guanosine-diphosphatase
305 EC 3 .6.1.45 UDP-sugar diphosphatase
306 EC 3 .6. 1.53 Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase
307 EC 3 .6.2.1 adenylylsulfatase
308 EC 3 .6.2.1 adenylylsulfatase
309 EC 3 .6.5.1 heterotrimeric G-protein GTPase
310 EC 3 .6.5.2 small monomeric GTPase
31 1 EC 3 .6.5.3 protein-synthesizing GTPase
312 EC 3 .6.5.4 signal-recognition-particle GTPase No. IUBMC Enzyme Code & Name
313 EC 3.6. ,5.5 dynamin GTPase
314 EC 3.6. ,5.6 tubulin GTPase
315 EC 4.1. , 1.23 orotidine-5'-phosphate decarboxylase
316 EC 4.1. , 1.32 phosphoenolpyruvate carboxykinase (GTP)
317 EC 4.6. , 1.2 guanylate cyclase
318 EC 4.6. , 1.15 FAD-AMP lyase (cyclizing)
319 EC 6.2. .1.4 succinate— CoA ligase (GDP-forming)
320 EC 6.2. , 1 .6 glutarate— CoA ligase
321 EC 6.2. , 1.10 acid— CoA ligase (GDP-forming)
322 EC 6.3. ,2.5 phosphopantothenate— cysteine ligase
323 EC 6.3. ,4.4 adenylosuccinate synthase
324 EC 6.5. , 1.2 DNA ligase (NAD+)
Reaction Mixtures of the Invention
[00100] In addition to the methods and specific assays disclosed herein, the present invention provides reaction mixtures designed for practicing the disclosed methods and assays. These reaction mixtures can be configured differently, depending upon the specific assay to be conducted, however, the reaction mixtures must eventually include a purified recombinant NMPK in an appropriate buffer at an appropriate concentration, for catalyzing a particular NER required for conducting an assay of the invention. In these reaction mixtures the purified recombinant NMPK is chosen from AMPK, CMPK, GMPK, TMPK and UMPK according to nucleotide that is to be qualitatively or quantitatively analyzed, or the substrate requirements of the NTPase whose activity is to be assayed.
[00101] Optionally, the reaction mixtures may also contain Mg2+ in a
concentration appropriate for use as a required cofactor of the included NMPK. Also optionally, the reaction mixtures may also contain or one or more sufficiently pure nucleotides (i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate) to serve as a substrate (i. e. , phosphate acceptor or phosphate donor) as required for the NER to be catalyzed by the selected purified recombinant NMPK. [00102] The following represent a non-exhaustive list of reaction mixtures of the present invention, as configured for conducting specific methods (i.e. assays) of the invention.
[00103] For the detection and/or quantification of NMPs or dNMPs, the present invention provides a reaction mixture comprising:
(1) a cognate NMPK (i.e. , an NMPK having a phosphate acceptor specificity
matching that of the NMP or dNMP to be detected),
(2) a BTP that can either be from the same class of nucleotide as the NMP or dNMP to be detected, or preferably is from a different class of nucleotides, and
(3) ADP.
[00104] For the detection and/or quantification of NDPs or dNDPs, the present invention provides a reaction mixture comprising:
(1) a cognate NMPK (i.e. , an NMPK having a phosphate acceptor specificity
matching that of the NMP or dNMP to be detected), and
(2) ADP.
[00105] For the detection and/or quantification of NTPs other than ATP (i.e., BTPs), the present invention provides a reaction mixture comprising:
(1) an NMPK that can effectively utilize the BTP to be detected as a phosphate
donor, and that has a phosphate acceptor specificity that matches the NMP or dNMP to be included in the reaction,
(2) cognate NMP or dNMP (i. e. , matching the phosphate acceptor specificity of the NMPK to be used), and
(3) ADP.
[00106] For the detection and/or quantification of NTPase activity, the present invention provides a reaction mix comprising:
(1) a cognate NMPK capable of using the NDP generated by the activity of the
NTPase (i.e., GMPK for GDP) to produce ATP from ADP, and
(2) ADP.
[00107] As noted, these reaction mixtures may also optionally contain Mg2+, in a concentration appropriate for use as a required cofactor of the included NMPK. Also, the reaction mixtures may optionally contain or one or more sufficiently pure additional nucleotides (i.e. , nucleoside triphosphate, nucleoside diphosphate and/or nucleoside monophosphate) to serve as a substrate (i. e. , phosphate acceptor or phosphate donor) as required for the NER to be catalyzed by the included purified recombinant NMPK, or as required to amplify the signal (i. e. , ATP) generated by the NER through subsequent cycles of the NER.
[00108] Futher, the reaction mixtures may also optionally contain those reagents required for the ADR that is to be coupled to the NER. Such reagents may include, for example, luciferase enzyme and luciferin.
Kits of the Invention
[00109] The present invention provides kits designed to facilitate practicing the disclosed methods and assays. These kits can be configured in various ways, containing various sets of reagents, depending upon the specific method or assay to be conducted, or, alternatively, they can include a collection of reagents required to conduct a plurality of the methods and assays of the invention. Ideally the kits include a purified recombinant NMPK at an appropriate concentration, and in an appropriate buffer in a suitable resealable container, and (2) instructions for the use of the purified
recombinant NMPK to conduct a method or methods, or an assay or assays, of the invention, which have as a nexus the NER catalyzed by the included purified
recombinant NMPK. For such kits the NMPK is chosen from AMPK, CMPK, GMPK, TMPK and UMPK, depending upon the methods and assays that are to be conducted.
[00110] Optionally, the kits may also contain Mg2+ in water or in a suitable buffer, at a concentration appropriate for use as a required cofactor of the included NMPK, in a suitable container. Also optionally, the kits may also contain, in suitable containers, one or more solutions comprising a sufficiently pure nucleotide (i. e. , nucleoside triphosphate, nucleoside diphosphate or nucleoside monophosphate) for use either in either conducting a method or assay of the invention (i. e. , which are based upon the NER catalyzed by the included purified recombinant NMPK), or for use as a positive or negative control sample. Alternatively, the one or more sufficiently pure nucleotides may be provided in a dried or lyophilized form that can be rehydrated, resuspended or dissolved by the end user to form a solution, or solutions, for use in the methods and assays of the invention.
[00111] Also optionally, the kits may also contain one or more suitable containers containing a solution comprising a cyclic nucleotide phosphodiesterase, for use in assays designed to detect or quantify a particular cyclic nucleotide in a sample, as shown in Figure 6. Optionally, the kits may also contain one or more suitable containers containing a solution comprising a GTPase to be used as a positive control in assays of GTPase activity, such as those depicted in Figures 7 and 8, or described in Example 4 or 5, below.
[00112] In some embodiments, the kits of the invention also contain reagents sufficient for conducting the ADR, which is to be coupled to the NMPK-catalyzed NER that the kit is designed to facilitate. As noted above, any suitable ADR can be utilized for the assays of the present invention, so long as the ADR can be coupled to the NER, and so long as the ADR consumes or otherwise eliminates the ATP generated by the NER. In particular embodiments of the invention the ADR involves the generation of light by the reaction catalyzed by ATP-dependent luciferase. In these embodiments the kits of the present invention can comprise the reagents required to conduct the ADR, including purified recombinant luciferase and luciferin.
[00113] One particular set of embodiments involve the use of firefly luciferase, and its substrates, luciferin and 02, in the ADR to detect the ATP generated by an NER of the present invention. One commercial embodiment along these lines is the
ATPLite™ system, which is available from PerkinElmer of Waltham, MA. Details of the specific reagents and reactions at the core of the ATPLite™ system are found in U.S. Patent 6,503,723, the contents of which are incorporated by reference herein in their entirety.
[00114] In view of the suitability and sensitivity of the ATPLite™ system when used as the ADR coupled to the NER of the assays of the present invention - as demonstrated in the examples below - in certain embodiments, the kits of the present invention particularly comprise and contain the reagents of the PerkinElmer ATPLite™ system, or the reagents taught in U.S. Patent 6,503,723. [00115] In some embodiments the kits of the present invention contain some or all of the reagents required to conduct a particular methods or assay of the invention in some type of carrier or compartmentalized container. In other words, the kits of the invention contain some or all of the reagents required to conduct a particular NER and coupled ADR, in some type of carrier or compartmentalized container. Ideally, the reagents included in the kits are contained within appropriate resealable containers. Ideally such containers are included in the kits in specific locations of the carrier or compartmentalized container. The choice of specific location for a give reagent should ideally create some degree of organization or orchestrated grouping of the reagents. For example, all of the reagents required for the NER may be grouped in one group, while the reagents required for the ADR may be grouped in a second group.
[00116] The carrier or compartmentalized container can comprise a container, vessel, or support, in the form of, e.g. , a bag, box, foam block, or rack, that is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage, and such a carrier may be designed to enclose the compartmentalized container. The carrier or compartmentalized container may ideally be designed such that the components carried therein remain in an organized state during shipment and storage. Finally, the carrier optionally contains instructions for the use of the contents of the carrier in conducting one or more of the methods and assays of the present invention.
EXAMPLES
[00117] The following examples are illustrative, but are not intended to limit the scope of the possible embodiments of the present invention. Any suitable modifications and adaptations of the variety of conditions, reagents and parameters normally encountered in the practice of biochemical assays, and which would be understood by those skilled in the art, should be understood to be within the spirit and scope of the invention. Example 1 : Cloning, Overexpression & Purification of AMPK, CMPK, GMPK and
UMPK from Escherischia coli.
[00118] The Escherischia coli K- 12 strain, MG1655 {i. e. , DH5 ) substrain, possesses five different NMPK-encoding genes. These genes have been given different names as a result of multiple naming conventions. The table below provides: (a) the names of the different NMPK genes associated with the NMPK they encode; (b) the EcoGene Accession Numbers for these genes (as provided by the EcoGene database maintained by the University of Miami and available via the internet at
www.ecogene.org), and (c) their respective coding regions as provided within the reference E. coli genomic sequence given in Entrez Nucleotide listing U00096.2 {i. e. , GI: 48994873).
Table 1
Figure imgf000041_0001
[00119] The Nucleotide Monophosphate Kinases AMPK, CMPK, GMPK, TMPK and UMPK were cloned from the K- 12 DH5 or DH10B strain of E. coli using routine methods that are well-known to artisans skilled in the art of cloning, expression and purification of recombinant bacterial proteins. In general, specifically designed pairs of primers were used to amplify the coding regions of the five NMPKs (identified in Table 1) from isolated K- 12 DH5 genomic DNA using the Polymerase Chain Reaction (PCR). E. Coli K-12 strain DH10B or DH5a cells were heat treated to obtain a simple lysate, and this lysate was used as the source of template genomic DNA from which the NMPK coding regions were amplified using the different NMPK-specific primer pairs. The primer pairs were designed to facilitate the specific amplification of a particular NMPK coding region, and were also designed to facilitate cloning of the amplified DNA.
[00120] Amplified DNAs (i. e. , PCR products) encoding the recombinant NMPKs, GMPK, CMPK, AMPK, UMPK, and TMPK were gel purified (Figs. 9A, 10A, 1 1A, 12A, and 13) and cloned, in-frame, into a modified pET expression vector that included an upstream initiation codon followed by six in-frame histidine codons, under the control of a T7 promoter. The completed expression vectors comprised a translation initiation codon encoding the N-terminal methionine, followed by six in-frame histidine codons that were, in-turn, positioned in frame with the native initiation methionine and subsequent coding region for the particular NMPK. The six in-frame histidine codons served to add an N-terminal "hexa-histidine tag" (i.e. , "His6 tag") to the expressed protein. This His6 tag facilitated rapid purification of recombinant protein as described below. DNA sequencing was used to confirm that the sequence encoding the NMPK was properly inserted in-frame with the N-terminal His6 tag to create the desired expression vector.
[00121] The completed expression vectors were transformed into either the BL21 DE3 or BL21 AI strain, and expression of the encoded recombinant NMPK with N- terminal His6 tag, was induced with either IPTG for the transformed BL21 DE3 strain, or IPTG and L-arabinose for the transformed BL21 -AI strain. Cell densities and induction times required for maximal expression were determined on small batches of transformed cells, and expressions for purification were scaled up accordingly.
[00122] Transformed expression strains were grown to appropriate densities and induced to overexpress a particular recombinant His-tagged NMPK, for a desired period of time. Cells were isolated by centrifugation and lysed. Lysates were applied to Ni2+- agarose columns in order to isolate the recombinant His-tagged NMPK by immobilized metal ion affinity chromatography. The His6-tagged, recombinant NMPKs adhered to the column, while the majority of other cellular proteins eluted from the column. After a wash step, the bound His-tagged, recombinant NMPKs were eluted from the column using a gradient of increasing concentrations of imidizole (Figs. 9B, 10B, 1 1B, & 12B). Isolated fractions were collected and analyzed by SDS-PAGE (Figs. 9B & 10B).
Fractions containing the highest concentrations of recombinant NMPKs were pooled and dialyzed to prepare stock solutions of the different NMPKs.
[00123] By this method, His-tagged recombinant E. Coli NMPKs were isolated to high levels of purity {i. e. , 90% or greater), and yields from about 100 to 400 mg of recombinant protein per liter of cultured transformed E. coli were obtained.
Representative amplification steps for GMPK, CMPK, AMPK, UMPK, and TMPK are show in Figures 9A, 10A, 1 1A, 12A, and 13, respectively. Representative purification steps for GMPK, CMPK, AMPK, and UMPK are show in Figures 9B, 10B, 1 1B, and 12B, respectively.
[00124] Four recombinant E. Coli NMPKs {i. e. , His6-GMPK, His6-CMPK, His6- AMPK, and His6-UMPK) obtained by the above-described procedures were used to catalyze the representative Nucleotide Exchange Reactions described in the following examples.
Example 2: Effect of GDP and GTP on GMPK-Catalyzed Nucleotide Exchange
Reactions
[00125] To assess the effect of GDP and GTP on GMPK-catalyzed NERs, a GMPK-catalyzed NER was coupled with a luciferase-catalyzed ATP detection reaction (ADR) as schematically shown in Figure 2. The basic reactions were set up as follows: A 100 μΐ total volume sample (containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 μΜ ADP, +/- 10 μΜ GTP, +/- 0.1 μΜ GMPK) is titrated with increasing concentrations of GDP from 0.005 μΜ to 5 μΜ. The reaction was incubated at room temperature for 30 min. whereupon 50 μΐ of ADR solution (ATPLite™ solution; PerkinElmer; Waltham, MA) was added to the samples and the luminescent signal measured with a Topcount plate reader (PerkinElmer; Waltham, MA).
[00126] The reaction mixture contained 10 mM Mg2+, 10 μΜ ADP, 10 μΜ GTP, 0.1 μΜ GMPK, and increasing concentrations of GDP from 0.005 to 5 μΜ. Control reactions were conducted in which GTP, GMPK, or both, were left out. The results are shown in Figure 14. [00127] It should be noted that, at sufficient levels, GDP can be readily detected and quantified when the same reaction is conducted in the absence of GTP. However, addition of GTP to the reaction mixture results in the amplification of the signal generated, due to the cyclic nature of the NER. This assay was found to be highly sensitive and can be used to determine concentrations of GDP as low as 0.05 μΜ.
These results are also shown in Figure 14.
Example 3: Detection and Quantification of GDP Using GMPK-Catalyzed
Nucleotide Exchange Reactions
[00128] The basic reactions were set up as follows: A 100 μΐ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, as well as the components indicated in Fig. 15. Samples were incubated at room temperature for 30 min, then 50 μΐ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was added to the samples. Luminescence signal was measured with a Topcount plate reader
(PerkinElmer; Waltham, MA).
[00129] The data shown in Figure 15 clearly demonstrate light generated by the coupled NER and ADR reactions is dependent upon the presence of GDP (G). As noted above, addition of GTP to the reaction results in an amplification of the bioluminescent signal (E). The small amount of light generated when ADP and GTP are included in the reaction but GDP is excluded (I) may result from a small amount of GDP contamination in the GTP stock, or alternatively, from a small amount of spontaneous hydrolysis of the GTP to GDP and Pi. However, the amount of light produced in the absence of GMPK (F & H) shows that the NER is clearly driving the generation of light by the ADR.
Example 4: Quantification of DLP1 GTPase Activity Using GMPK-Catalyzed
Nucleotide Exchange Reactions
[00130] DLP 1 (also known as dynamin 1 -like; dynamin-like protein 4; dynamin- like protein IV; Dnmlp/Vpslp-like protein; and dynamin-related protein 1 ; and encoded by Entrez GenelD: 10059) is a member of the dynamin superfamily of GTPases.
Members of the dynamin-related subfamily, including the S. cerevisiae proteins Dnml and Vps l , contain the N-terminal tripartite GTPase domain but do not have the pleckstrin homology or proline-rich domains. This protein establishes mitochondrial morphology through a role in distributing mitochondrial tubules throughout the cytoplasm.
[00131] The methods of the present invention were utilized to develop an assay for the GTPase activity of recombinant DLP l . The assay utilized a GMPK-catalyzed NER coupled with a luciferase-catalyzed ADR to detect the GDP produced as a result of GTP hydrolysis catalyzed by DLP l , as shown schematically in Figure 7. The activity of wild type DLPl was compared to that of a catalytically-deactivated mutant form, in which a key lysine residue (K38) was substituted with an alanine. This "K38A" mutant form of DLP l was used a negative control.
[00132] Experiments using a wild type DLP l and catalytically inactive K38A DLP l are shown in Fig. 16. The 75 μΐ total volume samples contained 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 10 μΜ GTP, and 0.2-1 μΜ DLP l WT or DLP (K38A) mutant protein as indicated. Reactions were performed at room temperature for 50 minutes, whereupon ADP and GMPK were added to final a concentration of 15 μΜ and 0.1 μΜ, respectively. Samples were incubated at room temperature for 10 min. and 50 μΐ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was then added to the samples. Luminescence signal was measured with a Topcount plate reader
(PerkinElmer; Waltham, MA). For other experiments in Fig. 16, 100 μΐ total volume samples (containing 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 15 μΜ ADP, 0.1 μΜ GMPK as well as other components as indicated) were incubated at room temperature for 30 min. 50 μΐ of ADR solution (ATPLite solution) was then added to the samples and the luminescent signal was measured with a Topcount plate reader.
[00133] Figure 16 shows the results of the assay conducted with three different concentrations of wild type or K38A DLP. Increasing concentrations (0.2, 0.5 and 1.0 μΜ) of wild type DLPl resulted in a substantial increase in the amount of light generated. In contrast increases in concentrations (0.2, 0.5 and 1.0 μΜ) of the K38A mutant form of DLP l resulted in a slight increase in the amount of light generated. This slight increase in light may be due to the K38A form of DLPl having a very small amount of catalytic activity, relative to the wild type enzyme. [00134] Control reactions in which no DLP l was added, but in which ADP, GTP, GDP, GDP+GTP, or ATP was added, show the expected results; namely, that the coupled reactions were dependent upon the presence of GDP. Again, the small amount of light generated when ADP and GTP are included in the reaction but GDP is excluded (I) may be a result of a small amount of GDP contamination in the GTP stock, or may result from the spontaneous hydrolysis of the GTP to GDP and Pi.
Example 5: High Throughput Screening (HTS) of DLPl GTPase Activity Using
GMPK-Catalyzed Nucleotide Exchange Reactions
Principle of the assay:
[00135] GMPK-catalyzed nucleotide exchange reactions can be used for high throughput screening of compounds which inhibit DLPl enzymatic activity. Solution compositions to be used are as indicated below. Mixing 30 μΐ Solution A and 10 μΐ Solution B results in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.01 % (w/v) Triton X- 100, 10 μΜ ADP, 10 μΜ GTP, and 0.5 μΜ DLP l . 0.5 μΐ of compound or vehicle alone (as a negative control) was then added to the samples and incubated at room temperature for 1 - 1.5 hours. After incubation, 10 μΐ of Solution D (50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 0.01 % Triton X- 100, 0.4 μΜ GMPK and 50% ATPLite solution) was added and further incubated at room temperature for 30 min. The resulting luminescent signal generated was measured with a Topcount plate reader (PerkinElmer; Waltham, MA). The "Counter Screen" is a compound specificity control, in which DLP l was omitted and 10 μΜ GDP was added. High throughput screen performance was evaluated using the same procedures as the described above, in the presence and absence of DLPl . Results are shown in Fig. 17.
[00136] The goal of this experiment was to assess the suitability of the coupled GMPK-catalyzed NER and ADR for use in a high-throughput format. In the replicate reactions tested, DLP l -catalyzed production of GDP is coupled to light generation by a luciferase-based ADR, as depicted in Figure 7.
[00137] The reactions were conducted as follows:
Figure imgf000047_0001
Figure imgf000048_0001
Working solutions:
TTMg buffer:
50 mM Tris-HCl pH 7.5
10 mM MgCl2
0.01 % (w/v) Triton X- 100
Can be stored w/o Triton X- 100 at 4°C for up to a week; add Triton X- 100 immediately before use.
Solution A:
13.3 μΜ GTP
13.3 μΜ ADP
in TTMg
Prepared immediately before use.
Solution B:
2 μΜ DLP 1 in TTMg
Prepared immediately before use.
Solution C:
40 μΜ GDP in TTMg
Prepared immediately before use.
Solution D:
0.4 μΜ GMPK
50%) ATPlite substrate solution (lyophilized substrate + substrate buffer) (PerkinElmer; Waltham, MA) in TTMg
Prepared immediately before use.
Procedure:
Use Corning Costar (Lowell, MA) 96-well half area plate (cat # 3694) for the assay.
Figure imgf000049_0001
Analyst AD Setting:
Mode: luminescence
Plate format: Greiner 96 flat black PS
Luminescence height: 1 mm
Readings per well: 5
Integration time: 100,000
Units: RLU
HTS Assay Fitness Parameters:
1>Z'>0.9: excellent
0.9>Z'>0.7: good
0.7>Z'>0.5 : acceptable
Z'=0.5 : minimum
Calculated HTS Assay Fitness:
[00138] The results of the HTS assay are shown in Figure 17.
[00139] As noted above, the reaction mixture contains Tris buffer, 10 mM MgCl2, 10 μΜ ADP, 10 μΜ GTP, 0.1 μΜ GMPK. 0.4 μΜ wild type DLP l (upper set of data points). Omitting DLP l from the mixture is designed as background (lower set of data points). ATP generated from the reaction is measured with commercially available luciferase based ATPlite solution (PerkinElmer, Cat # 6016949; Waltham, MA).
[00140] This standard HTS performance evaluation assay indicates that the NER based DLP l GTPase activity assay has excellent HTS performance quality (Z' factor) and excellent signal background and signal:noise ratios. Example 6: Detection and Quantification of CMP Using CMPK-Catalyzed
Nucleotide Exchange Reactions
[00141] The basic reactions were set up as follows: 100 μΐ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 and the components indicated in Figure 18 (the numbers along the x axis indicate the concentrations of nucleotides, in μΜ). 0.1 μΜ CMPK was present or absent as indicated. Samples were incubated at room temperature for 30 min, whereupon 50 μΐ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was added and the luminescent signal measured with a Topcount plate reader.
[00142] A CMPK-catalyzed NER coupled to a luciferase-catalyzed ADR, as depicted in Figure 18A, was used to detect the presence of CMP in a sample. The phosphate donor employed in the forward reaction of the NER was GTP. The use of GTP, rather than CTP, is made possible by the relatively relaxed substrate specificity of E. coli CMPK for the phosphate-donating nucleotide. The results (Figure 18B) clearly demonstrate that the assay is CMP-dependent, and show that the use of GTP as the phosphate donor in the forward reaction works well, with only a slight background signal being generated.
Example 7: Detection and Quantification of AMP and ADP Using AMPK- Catalyzed Nucleotide Exchange Reactions
[00143] The basic reactions were set up as follows: 80 μΐ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgC12 and the components indicated in Figure 19 (the numbers along the x axis indicate the concentrations of nucleotides, in μΜ). 0.34 μΜ AMPK was present or absent as indicated. Samples were incubated at room temperature for 60 min, whereupon 40 μΐ of ADR solution (ATPLite solution; PerkinElmer; Waltham, MA) was added and the luminescent signal was measured with a Topcount plate reader (PerkinElmer; Waltham, MA).
[00144] As noted previously, AMPK can catalyze AMP phosphorylation with dATP as a phosphate donor, to produce ADP. Thus, no exogenous ADP is needed for the AMPK-catalyzed NER in order to detect and quantify levels of AMP. In the "reverse reaction" of the NER one molecule of ADP can act as a phosphate donor for the AMPK-catalyzed phosphorylation of another molecule of ADP, thereby generating ATP and AMP. The ATP generated by this reaction can then be detected by a coupled ADR, while the AMP can be reused for the cycling reaction, thereby amplifying the signal produced.
[00145] To demonstrate the utility of this approach, an AMPK-catalyzed NER coupled to a luciferase-catalyzed ADR, as depicted in Figure 19A, was used to detect the presence of AMP alone, or ADP alone, in a sample. The phosphate donor employed in the forward reaction of the NER was dATP. The use of dATP, rather than ATP (or GTP), is made possible by the relatively relaxed substrate specificity of E. coli AMPK for the phosphate-donating nucleotide. Importantly, dATP is not a substrate for the luciferase-catalyzed ADR, and results in only a small background signal. The results (Figure 19B) clearly demonstrate that the assay is either AMP- or ADP-dependent; that the use of dATP as the phosphate donor in the forward reaction works well; and that the signal generated by the coupled reactions is proportional to the amount of AMP or ADP in the added sample.
Example 8: Detection and Quantification of UMP and UDP Using UMPK- Catalyzed Nucleotide Exchange Reactions
[00146] The basic reactions were set up as follows: 80 μΐ total volume samples contain 50 mM Tris-HCl (pH 7.5), 10 mM MgC12 and the components indicated in Figure 20(the numbers along the x axis indicate the concentrations of nucleotides, in μΜ). 0.26 μΜ UMPK was present or absent as indicated. Samples were incubated at room temperature for 60 min, whereupon 40 μΐ of ADR solution (ATPLite solution) was added and the luminescent signal was measured with a Topcount plate reader.
[00147] In a manner similar to AMPK (as described in Example 7, above) UMPK can catalyze UMP phosphorylation using dATP as a phosphate donor, to produce UDP. In the "reverse reaction," UDP can act as a phosphate donor for the UMPK-catalyzed phosphorylation of exogenous ADP, to generate ATP and UMP. The ATP generated by this reaction can then be detected by a coupled ADR, while the UMP can be reused for the cycling reaction, thereby amplifying the signal produced. [00148] To demonstrate the utility of this approach, a UMPK-catalyzed NER coupled to a luciferase-catalyzed ADR, as depicted in Figure 20A, was used to detect the presence of UMP alone, or UDP alone, in a sample. The phosphate donor employed in the forward reaction of the NER was dATP. The use of dATP, rather than UTP (or GTP), is made possible by the relatively relaxed substrate specificity of E. coli UMPK for the phosphate-donating nucleotide. Importantly, dATP is not a substrate for the luciferase-catalyzed ADR, and results in a very small background signal. The results (Figure 20B) clearly demonstrate that the assay is UMP- or UDP-dependent, that the use of dATP as the phosphate donor in the forward reaction works well, and that the signal generated by the coupled reactions is proportional to the amount of UMP or UDP in the added sample.
[00149] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which the claimed invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. However, the mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.
[00150] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to the skilled artisan that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A process comprising a first reaction coupled to a second reaction, wherein:
said first reaction is catalyzed by a nucleoside monophosphate kinase (NMPK) and generates adenosine triphosphate (ATP); and
said second reaction detects the ATP generated by said first reaction.
2. The process of claim 1 , wherein said NMPK acts to transfer a phosphate group from a phosphate donor to a phosphate acceptor and wherein said NMPK acts to further transfer a phosphate group to adenosine diphosphate (ADP) to generate ATP.
3. A method of detecting the presence of a nucleotide in a sample comprising:
utilizing the process of claim 1 , wherein said nucleotide is a nucleotide other than ATP, wherein said NMPK of the first reaction is capable of utilizing said nucleotide as a substrate, wherein if said nucleotide is present in said sample, then ATP is generated by said first reaction and then detected by said second reaction, wherein detection of generated ATP by said second reaction indicates said nucleotide is present in said sample.
4. The method of claim 3, further comprising quantifying the amount of generated ATP, wherein the amount of generated ATP indicates the amount of said nucleotide in said sample.
5. The method of claim 3, further comprising identifying an NMPK that phosphorylates said nucleotide.
6. A reaction mixture for carrying out the process of claim 1 for detecting the presence or amount of a GTPase present in a sample, said reaction mixture comprising:
(a) GTP; (b) a guanosine monophosphate kinase (GMPK);
(c) ADP; and
(d) optionally, a nucleoside triphosphate other than ATP or GTP, capable of use as a phosphate donor by the GMPK included in the reaction mixture.
7. A reaction mixture for carrying out the process of claim 1 for detecting the presence or amount of a particular nucleoside monophosphate (NMP) present in a sample, said reaction mixture comprising:
(a) an NMPK capable of utilizing, as a phosphate acceptor, said particular NMP that is to be detected;
(b) an NTP, other than ATP, capable of use by the NMPK as a phosphate donor; and
(c) ADP.
8. A reaction mixture for carrying out the process of claim 1 for detecting the presence or amount of a particular nucleoside diphosphate (NDP) present in a sample, said reaction mixture comprising:
(a) an NMPK capable of utilizing, as a phosphate donor, the particular NDP that is to be detected; and
(b) ADP.
9. A reaction mixture for carrying out the process of claim 1 for detecting the presence or amount of a particular nucleoside triphosphate (NTP), other than ATP, present in a sample, said reaction mixture comprising:
(a) an NMPK capable of utilizing, as a phosphate donor, the NTP, other than ATP, that is to be detected;
(b) a nucleoside monophosphate capable of use as a phosphate acceptor by the NMPK; and
(c) ADP.
10. A reaction mixture for carrying out the process of claim 1 for detecting the presence or amount of a particular cyclic nucleoside monophosphate (cNMP) present in a sample, said reaction mixture comprising:
(a) a cyclic nucleotide phosphodiesterase capable of converting said particular cNMP to a 5 'phosporylated corresponding NMP;
(b) an NMPK capable of utilizing, as a phosphate acceptor, the particular 5 'phosporylated corresponding NMP generated by said cyclic nucleotide
phosphodiesterase;
(c) an NTP, other than ATP, that can be used by the NMPK as a phosphate donor; and
(d) ADP.
1 1. A method for quantifying the amount or activity of a GTPase present in a sample, said method comprising combining said sample with:
(a) GTP;
(b) a purified GMPK;
(c) ADP; and
(d) optionally, an NTP, other than ATP or GTP, capable of use as a phosphate donor by the GMPK;
detecting any ATP generated, wherein the amount of any ATP generated is indicative of the amount or activity of the GTPase present in said sample.
12. The method of claim 1 1 , wherein said NTP, other than ATP or GTP, capable of use by the NMPK as a phosphate donor, is dATP or a non-cognate NTP, other than ATP.
13. The method of claim 1 1 , wherein said any ATP generated is detected using a coupled ATP detection assay.
14. The method of claim 13, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
15. A method for detecting the presence of a particular NMP present in a sample, said method comprising combining said sample with:
(a) an NMPK capable of utilizing, as a phosphate acceptor, the particular NMP that is to be detected;
(b) an NTP, other than ATP, capable of use by the NMPK as a phosphate donor; and
(c) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular NMP in said sample is indicated.
16. The method of claim 15, wherein the amount of said any ATP generated indicates the amount of the particular NMP present in the sample.
17. The method of claim 15, wherein said particular NMP is a ribonucleoside monophosphate (rNMP) or a deoxyribonucleoside monophosphate (dNMP).
18. The method of claim 15, wherein said NTP, other than ATP, capable of use by the NMPK as a phosphate donor is dATP or a non-cognate NTP, other than ATP.
19. The method of claim 15, wherein said any ATP generated is detected using a coupled ATP detection assay.
20. The method of claim 19, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
21. A method for detecting the presence of a particular NDP present in a sample, said method comprising combining said sample with:
(a) a purified NMPK capable of utilizing, as a phosphate donor, the particular NDP that is to be detected; and (b) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular NDP in said sample is indicated.
22. The method of claim 21 , wherein the amount of said any ATP generated indicates the amount of the particular NDP present in the sample.
23. The method of claim 21, wherein said particular NDP is a ribomicleoside diphosphate (rNDP) or a deoxyribonucleoside diphosphate (dNDP).
24. The method of claim 21, wherein said NTP, other than ATP, capable of use by the NMPK as a phosphate donor is dATP or a non-cognate NTP, other than ATP.
25. The method of claim 21, wherein said any ATP generated is detected using a coupled ATP detection assay.
26. The method of claim 25, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
27. A method for detecting the presence of a particular NTP, other than ATP, present in a sample, said method comprising combining said sample with:
(a) a purified NMPK capable of utilizing, as a phosphate donor, the particular NTP that is to be detected;
(b) an NMP that can be used as a phosphate acceptor by the NMPK; and
(c) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular NTP, other than ATP, in said sample is indicated.
28. The method of claim 27, wherein the amount of said any ATP generated indicates the amount of the particular NTP, other than ATP, present in the sample.
29. The method of claim 27, wherein said particular nucleoside triphosphate is a ribonucleoside triphosphate (rNTP) or a deoxyribonucleoside triphosphate (dNTP).
30. The method of claim 27, wherein said any ATP generated is detected using a coupled ATP detection assay.
31. The method of claim 30. wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
32. A method for detecting the presence of a particular cNMP present in a sample, said method comprising combining said sample with:
(a) a cyclic nucleotide phosphodiesterase capable of converting said particular cNMP to a 5' phosporylated corresponding NMP;
Cb) a purified NMPK capable of utilizing, as a phosphate acceptor, the particular 5'phosporylated corresponding NMP generated by said cyclic nucleotide
phosphodiesterase;
(c) an NTP, other than ATP, capable of use by the NMPK as a phosphate donor; and
(d) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular cNMP in said sample is indicated.
33. The method of claim 32, wherein the amount of said any ATP generated indicates the amount of the particular cNMP present in the sample.
34. The method of claim 32, wherein said particular cyclic nucleoside monophosphate is a cyclic ribonucleoside monophosphate (c-rNMP) or a cyclic deoxyribonucleoside (c- dNMP) monophosphate.
35. The method of claim 32, wherein said NTP, other than ATP, capable of use by the NMPK as a phosphate donor is dATP or a non-cognate NTP, other than ATP.
36. The method of claim 32, wherein said any ATP generated is detected using a coupled ATP detection assay.
37- The method of claim 36, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
38. A method for detecting the presence of a particular nucleoside diphosphate present in a sample, said method comprising combining said sample with:
(a) a purified NMPK capable of utilizing, as a phosphate donoi, the particular nucleoside diphosphate that is to be detected; and
(b) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular nucleoside diphosphate in said sample is indicated.
39. The method of claim 38, wherein the amount of said any ATP generated indicates the amount of the particular nucleoside diphosphate present in the sample.
40. The method of claim 38, wherein said particular nucleoside diphosphate is a ribonucleoside diphosphate or a deoxyribonucleoside diphosphate.
41. The method of claim 38, wherein said nucleoside triphosphate, other than ATP, capable of use by the NMPK as a phosphate donor is dATP or a non-cognate NTP, other than ATP.
42. The method of claim 38, wherein said any ATP generated is detected using a coupled ATP detection assay.
43. The method of claim 42, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
44. A method for detecting the presence of a particular nucleoside triphosphate, other than ATP, present in a sample, said method comprising combining said sample with:
(a) a purified NMPK capable of utilizing, as a phosphate donor, the particular nucleoside triphosphate that is to be detected;
(b) a nucleoside monophosphate that can be used as a phosphate acceptor by the NMPK; and
(c) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular nucleoside triphosphate, other than ATP, in said sample is indicated.
45. The method of claim 44, wherein the amount of said any ATP generated indicates the amount of the particular nucleoside triphosphate, other than ATP, present in the sample.
46. The method of claim 44, wherein said particular nucleoside triphosphate is a ribonucleoside triphosphate or a deoxyribonucleoside triphosphate.
47. The method of claim 44, wherein said any ATP generated is detected using a coupled ATP detection assay.
48. The method of claim 47, wherein said coupled ATP detection assay is a coupled luciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled luciferase assay.
49. A method for detecting the presence of a particular cyclic nucleoside
monophosphate present in a sample, said method comprising combining said sample with: (a) a cyclic nucleotide phosphodiesterase capable of converting said particular cyclic nucleoside monophosphate to a 5'phosporylated corresponding nucleoside monophosphate;
(b) a purified NMPK capable of utilizing, as a phosphate acceptor, the particular 5'phosporylated corresponding nucleoside monophosphate generated by said cyclic nucleotide phosphodiesterase;
(c) a nucleoside triphosphate, other than ATP, capable of use by the NMPK as a phosphate donor; and
(d) ADP;
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular cyclic nucleoside monophosphate in said sample is indicated.
50. The method of claim 49, wherein the amount of said any ATP generated indicates the amount of the particular cyclic nucleoside monophosphate present in the sample.
51. The method of claim 49, wherein said particular cyclic nucleoside monophosphate is a cyclic ribonucleoside monophosphate or a cyclic deoxyribonucleoside
monophosphate.
52. The method of claim 49, wherein said nucleoside triphosphate, other than ATP, capable of use by the NMPK as a phosphate donor is dATP or a non-cognate NTP, other than ATP.
53. The method of claim 49, wherein said any ATP generated is detected using a coupled ATP detection assay.
54. The method of claim 53, wherein said coupled ATP detection assay is a coupled Iuciferase assay and the amount of said any ATP generated is proportional to the amount of light generated by said coupled Iuciferase assay.
55. A method of screening test compounds for their ability to inhibit a particular GTPase present in a sample, said method comprising combining said sample with:
(a) a test compound;
(b) GTP;
(c) a purified guanosine monophosphate kinase (GMPK);
(d) ADP; and
(e) optionally, an NTP, other than ATP or GTP, capable of use as a phosphate donor by the GMPK;
detecting any ATP generated, wherein the amount of ATP generated is indicative of the amount or activity of the particular GTPase present in said sample, and a reduction of the ATP generated in the presence of said test compound, relative to the amount generated in the absence of said test compound, indicates that said test compound inhibits said particular GTPase.
56. A kit comprising:
(a) an NMPK;
(b) an NTP, other than ATP, capable of use by the NMPK as a phosphate donor; and
(c) ADP;
in a compartmentalized container.
57. The kit of claim 56, further comprising instructions for using the kit.
58. A method of detecting the presence of a nucleotide, other than ATP, in a sample comprising:
combining (a), (b). and (c) of the kit of claim 56 or 57 with said sample; and determining whether any ATP is generated, wherein ATP generation indicates the presence of said nucleotide.
59. A method of detecting the presence of a nucleotide, other than ATP, in a sample comprising:
combining said sample with:
(a) an NMPK capable of phosphorylating said nucleotide,
(b) a phosphate donor other than ATP, and
(c) ADP; and
detecting any generation of ATP, wherein detection of ATP indicates the presence of said nucleotide in said sample.
60. The method of claim 59, further comprising detecting background levels of ATP in said sample.
61. The method of claim 60, wherein said detecting background levels step is performed before combining said NMPK with said sample.
62. A method for quantifying the amount or activity of a GTPase present in a sample, said method comprising:
combining said sample with the reaction mixture of claim 6; and
detecting any ATP generated, wherein the amount of any ATP generated is indicative of the amount or activity of the GTPase present in said sample.
63. A method for detecting the presence of a particular NMP present in a sample, said method comprising:
combining said sample with the reaction mixture of claim 7; and
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular NMP in said sample is indicated.
64. A method for detecting the presence of a particular NDP present in a sample, said method comprising:
combining said sample with the reaction mixture of claim 8; and detecting any ATP generated, wherein if ATP is generated, then, the presence of the particular NDP in said sample is indicated.
65. A method for detecting the presence of a particular NTP, other than ATP, present in a sample, said method comprising:
combining said sample with the reaction mixture of claim 9; and
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular NTP, other than ATP, in said sample is indicated.
66. A method for detecting the presence of a particular cNMP present in a sample, said method comprising:
combining said sample with the reaction mixture of claim 10; and
detecting any ATP generated, wherein if ATP is generated, then the presence of the particular cNMP in said sample is indicated.
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