EP1869083A1 - Procede d analyse destine aux reactions a transfert de groupe - Google Patents

Procede d analyse destine aux reactions a transfert de groupe

Info

Publication number
EP1869083A1
EP1869083A1 EP05785285A EP05785285A EP1869083A1 EP 1869083 A1 EP1869083 A1 EP 1869083A1 EP 05785285 A EP05785285 A EP 05785285A EP 05785285 A EP05785285 A EP 05785285A EP 1869083 A1 EP1869083 A1 EP 1869083A1
Authority
EP
European Patent Office
Prior art keywords
donor
product
tracer
assay
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05785285A
Other languages
German (de)
English (en)
Inventor
Robert Lowery
Karen Kleman-Leyer
Matt Staeben
Thane Westermeyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BellBrook Labs LLC
Original Assignee
BellBrook Labs LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BellBrook Labs LLC filed Critical BellBrook Labs LLC
Publication of EP1869083A1 publication Critical patent/EP1869083A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to group transfer reaction methodologies.
  • the invention provides methods for the detection and quantification of donor-products and the catalytic activities generating the donor-products in group transfer reactions.
  • the invention also provides methods for high throughput screening to identify acceptor substrates, inhibitors, or activators of enzymes catalyzing group transfer reactions.
  • the invention further provides immunoassays, antibodies and related kits for practicing the invention.
  • donor-X the activated donor molecule
  • the donor-X is a nucleotide attached to a covalent adduct.
  • the donor-X is activated by formation of a phosphoester bond in the nucleotide donor.
  • the acceptor substrates can include small molecules or macromolecules such as proteins or nucleic acids. Products of this reaction are the modified acceptor, acceptor- X and the donor-product molecule.
  • kinases which use ATP to donate a phosphate group
  • sulfotransferases which use phosphoadenosine-phosphosulfate (PAPS) to donate a sulfonate group
  • UDP- glucuronosyltransferases UDP-glucuronic acid to transfer a glucuronic acid group
  • methyltransferases which use s-adenosyltransferase to donate a methyl group
  • acetyl transferase which use acetyl coenzymeA to donate an acetyl group
  • ADP- ⁇ ribosyltransferases which use nicotinamide adenine dinucleotide (NAD) to donate an ADP- ribose group.
  • NAD nicotinamide adenine dinucleotide
  • HTS high throughput screening
  • UGTs are currently assayed using radiolabeled donor molecules and require post-reaction separation steps such as thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC) which seriously hampers preclinical HTS programs (Ethell, B. T., et al., Anal Biochem, 1998, 255:142-7).
  • TLC thin layer chromatography
  • HPLC high pressure liquid chromatography
  • kinases have been assayed by filter capture or precipitation of radiolabeled polypeptide substrates produced using 32 P- ATP or 33 P-ATP as donor.
  • this method requires a separation step such as filtering or centrifugation, it cannot be easily adapted to an automated HTS format.
  • SPA Surface proximity assays
  • phosphorylation of substrate peptide leads to displacement of a fluorescent phosphopeptide tracer from an anti-phosphopeptide antibody and causes a change in its fluorescence properties.
  • This basic approach has been adapted to several different readout modes used for competitive immunoassays including Fluorescence polarization (FP) (Parker, G. J., et al., J Biomol Screen, 2000, 5:77-88); time resolved fluorescence (Xu, K., et al., J Biochem MoI Biol, 2003, 36:421-5); fluorescence lifetime discrimination (Fowler, A., et al., Anal Biochem, 2002, 308:223-31); and chemiluminescence (Eglen, R. M. and Singh, R., Comb Chem High Throughput Screen, 2003, 6:381-7).
  • FP Fluorescence polarization
  • the non-generic nature of the current group transfer assays is resulting in significant expense and delays for drug discovery because of the need to develop assays for individual enzymes or small subgroups within a family. Also, because many of the current assays are based on modification and detection of specific tagged acceptors, there is limited ability for testing different acceptor substrates. Often the tagged acceptor substrates used are different from the substrates that are phosphorylated in vivo, thus the physiological relevance of the assay is questionable. In addition, a major concern in the pharmaceutical industry is that because of the non-generic nature of the current assays, investigators are sometimes forced to use different methods for different kinases.
  • the assay products can be detected using homogenous fluorescence or chemiluminescence methods which are not subject to significant interference or background signal from molecules in pharmaceutical drug libraries.
  • the present invention is summarized as methods and components thereof for detecting the activity of and screening acceptor substrates, inhibitors, or activators of enzymes catalyzing group transfer reactions to facilitate the development of more selective and therapeutic drugs. This is accomplished through a highly selective antibody used to bind the donor product of the group transfer reaction. Antibody-antigen interactions can be detected in a number of ways that have already been described by others.
  • the detection mode applicants have used to put the method into practice is a competitive fluorescence polarization immunoassay (FPIA), because it is well suited for pharmaceutical HTS assays.
  • FPIA competitive fluorescence polarization immunoassay
  • enzymatically generated donor product displaces a fluorescent derivative of the donor product, called a tracer, from an antibody resulting in a decrease in tracer fluorescence polarization.
  • the key reagents for the assay are an antibody that binds with high selectivity to the donor product, and not to the uncleaved donor molecule, and a tracer - a fluorescent derivative of the donor product that retains its structure sufficiently to bind the antibody.
  • the invention provides a novel assay for detecting and quantifying activity for enzymes that catalyze group transfer reactions using diverse substrates.
  • the invention also provides a method of screening for substrates, inhibitors, or activators of the group transfer reactions.
  • the invention provides a method of detecting a donor-product of a group transfer reaction, the method including reacting an activated form of a donor with an acceptor in the presence of a catalytically active enzyme; forming the donor-product and an acceptor-X; contacting the donor-product with a first complex comprising a detectable tag capable of producing an observable; competitively displacing the detectable tag of the first complex by the donor product to generate a second complex and a displaced detectable tag; and detecting a change in the observable produced by the detectable tag in the first complex and the displaced detectable tag.
  • the invention provides an antibody produced against a donor product of a group transfer reaction, wherein the antibody has the ability to preferentially distinguish between a donor-product and a donor in the presence of a high donor concentration.
  • the antibody is generated using an immunogen made from a nucleotide conjugated to a carrier protein. The conjugation between the nucleotide and the carrier protein occurs at the N6 amino, C8 or C2 positions of adenine; the C5 or C6 positions of uridine, thymidine or cytidine; or the 2' or 3' OH of ribose.
  • the immunogen includes a linker region between the nucleotide and the carrier protein having a chemical formula of NH 2 -X-Z, wherein X is a saturated or unsaturated chain of 2 to 20 carbons and Z is a functional group capable of covalently binding to a protein, and wherein the group includes an NH 2 , SH, COOH or activated derivatives of COOH, such as succinimide or maleimide.
  • the invention provides a homogeneous competitive binding assay for a donor product of a group transfer reaction, the assay includes combining the donor- product with a tracer and a macromolecule to provide a mixture, the macromolecule being specific for the donor product, the tracer comprising the donor-product conjugated to a fluorophore, the tracer being able to bind to the macromolecule to produce a detectable change in fluorescence polarization; measuring the fluorescence polarization of the mixture to obtain a measured fluorescence polarization; and comparing the measured fluorescence polarization with a characterized fluorescence polarization value, the characterized fluorescence polarization value corresponding to a known donor-product concentration.
  • the invention provides assay kits for characterizing a donor- product from a group transfer reaction.
  • the assay kit includes a macromolecule and a tracer, each in an amount suitable for at least one homogeneous fluorescence polarization assay for donor-product, wherein the macromolecule is a an antibody or an inactivated enzyme.
  • the kit also includes a stabilizing reagent, preferably EGTA and a quenching reagent, preferably sodium vanadate.
  • the invention provides a tracer compound for use in a homogenous competitive binding assay to detect a donor-product of a group transfer reaction; wherein the tracer comprises a fluorophore conjugated to a nucleotide.
  • the fluorophore is conjugated to a nucleotide through a position selected from the group consisting of an N6 amino, a C8 or a C2 position of adenine, a C5 or C6 position of uridine, thymidine or cytidine, and a 2' or 3' OH position of ribose.
  • a linker may be positioned between the fluorophore and the nucleotide.
  • the linker includes a chemical formula OfNH 2 -X-Z; wherein X is a saturated or unsaturated chain of 2 to 20 carbons and Z is a functional group capable of covalently binding to a protein; and wherein the group includes an NH 2 , SH, COOH or activated derivatives of COOH, such as succinimide or maleimide.
  • the invention provides a chemical linker positioned between a fluorosphore and a nucleotide of a tracer compound; wherein the linker may include an aminoallyl, a diaminoalkane, or an aminoalkynyl group.
  • the invention provides a composition, preferably EGTA, for use in a homogenous competitive binding assay to stabilize an assay signal by preventing breakdown of a donor product.
  • the invention provides a composition, preferably sodium vandate, for use in a homogenous competitive binding assay to quench a group transfer reaction.
  • a composition preferably sodium vandate
  • FIG. 1 illustrates a fluorescence polarization immunoassay (FPIA) reaction for the SULT reaction product, PAP.
  • FIG. 2 illustrates use of FPIA to detect and quantify UDP formation, the donor product of the UGT reaction.
  • FIG. 3 illustrates use of FPIA to detect and quantify ADP formation, the donor product of the kinase reaction.
  • FIGS. 4 A-B depict examples of suitable tracers used to quantify UDP formation.
  • FIG. 5 shows titration or competitive displacement curves for uridine nucleotides using a polyclonal antibody raised against UDP/UTP and a commercially available tracer molecule (Alexa-UTP).
  • FIG. 6 shows standard UGT reaction conditions for detection of enzymatically generated UDP by competitive FPIA.
  • FIG. 7 shows the dependence of the FPIA-based UGT enzymatic assay on enzyme concentration and time.
  • FIG. 8 shows the dependence of the FPIA-based UGT enzymatic assay on acceptor concentration.
  • FIG. 9 shows use of the competitive FPIA for detection of UDP formation by diverse UGT isoforms and acceptor substrates.
  • FIGS. 10 A-B show competitive displacement of two different Alexa Fluor -UTP tracers from antibody by UDP and UDPGA.
  • FIG. 11 shows the effect of stabilizing reagents on the FPIA signal in UGT reactions.
  • FIG. 12 shows FP competition curves for ADP, ATP and AMP using a polyclonal antibody against ADP and a 5-FAM-N6-ADP tracer.
  • FIG. 13 shows synthesis of a 5-FAM-N6-ADP tracer.
  • FIG. 14 shows examples of tracers used to quantify ADP formation.
  • FIG. 15 shows the dependence of the FPIA-based kinase enzymatic assay on enzyme concentration and time.
  • FIG. 16 shows the synthesis of PAP antigens.
  • FIGS. 17 A-C show components of PAP tracer synthesis.
  • FIGS. 18 A-C show representative final PAP tracer structures.
  • FIG. 19 shows binding isotherms for anti-PAP antibodies and PAP- fluorescein tracers.
  • FIG. 20 shows competitive displacement of two different tracers from Ab 3642 by PAP and PAPS.
  • FIGS. 21 A-B show the competition curves with two Anti-PAP antibody/Tracer combinations.
  • FIG. 22 shows the effect of PAPS concentration on detection of enzymatically generated PAP.
  • FIGS. 23 A-F show a continuous FPIA-based detection of SULT activity with diverse substrates.
  • FIG. 24 shows a comparison of SULTlEl acceptor substrate profiles determined using the FPIA-based assay and the 35 S-PAPS radioassay.
  • FIG. 25 shows inhibition of SULTlEl by 2,6 Dichloro-4-nitrophenol (DCNP) measured with the FPIA-based assay.
  • the present invention broadly relates to novel assay methods for detecting and quantifying the donor-product or the catalytic activities generating the donor-product from group transfer enzymes using diverse substrates.
  • the invention also provides the antibodies specific to the donor product and assay kits for practicing the invention in a high-throughput screening format.
  • the general equation for the group transfer reaction includes a donor-X + acceptor -» donor-product + acceptor -X, wherein the donor-product is detected by the general detection reaction: first complex + donor-product -» second complex + displaced detectable tag.
  • a highly selective antibody is used to bind the donor product of the group transfer reaction, and this binding event is detected, using an immunoassay, such as for example a competitive binding FPIA which is well suited for pharmaceutical HTS assays.
  • an immunoassay such as for example a competitive binding FPIA which is well suited for pharmaceutical HTS assays.
  • enzymatically generated donor product displaces a tracer, from an antibody resulting in a decrease in tracer fluorescence polarization.
  • the key reagents for the assay are antibody that binds with high selectivity to the donor product, and not to the uncleaved donor molecule, and a tracer - a fluorescent derivative of the donor product that retains its structure sufficiently to bind the antibody.
  • the invention provides a universal assay method in that a single set of detection reagents can be used for all of the enzymes in a given family of group transfer enzymes and all acceptor substrates for that family.
  • This universal assay method provides a novel approach for accelerating the incorporation of SULTs, protein kinases and other group transfer enzymes into HTS screening.
  • the novel assay method would allow SULT isoforms (i.e., there are eleven known SULT isoforms) to be screened for their ability to sulfate diverse compounds in the same experiment using the same detection reagents, protocol and instrumentation.
  • This is an important capability because enzymes that catalyze xenobiotic conjugation (e.g., SULTs and UGTs) have very broad acceptor substrate specificity.
  • SULTs and UGTs enzymes that catalyze xenobiotic conjugation
  • kinases there is even more diversity within the enzyme family. There are over 400 protein kinases in humans, and there is great diversity in their acceptors substrate specificity such that either physiological protein acceptor substrates as well as short peptides may be used.
  • a number of protein kinases may be used in the assay of the invention using their diverse acceptor substrates and screened for inhibitors using the same detection reagents, protocol and instrumentation.
  • the FPIA based donor product detection assays of the invention for group transfer reactions such as SULT, UGT and kinases, among others are very well suited for automated HTS applications.
  • group transfer reaction refers to the general reaction: donor-X + acceptor ⁇ donor-product + acceptor-X.
  • Representative group transfer reactions are shown as follows:
  • Methyltransferase reaction s-adenosylmethionine + acceptor -> acceptor-CH 3 + s-adenosylhomocysteine;
  • Acetyl transferase reaction acetyl Coenzyme A + acceptor ⁇ acceptor-COCH 3 + CoenzymeA.
  • Group transfer reactions in-part such as sulfation by SULTs, phosphorylation by kinases and UDP-glucuronidation by UGTs are suitably applicable to the methods of the invention because one can isolate antibody/detectable tag pairs for the donor products, which are PAP, ADP and UDP, respectively.
  • group transfer reactions are generally involved in a number of biological processes, such as hormone biosynthesis and function; enzyme regulation and function; and xenobiotic metabolism.
  • universal assay and “generic assay” are used interchangeably to refer to a method whereby all members of the group transfer reaction enzyme family and all of their acceptor substrates can be detected with the same assay reagents.
  • group in group transfer reaction refers to the covalent adduct or the moiety that is being transferred from the donor molecule to the acceptor in a group transfer reaction.
  • Groups transferred in enzymatically catalyzed group transfer reactions typically include phosphates, sulfates, carbohydrates, naturally occurring amino acids, synthetically derived amino acids, methyls, acetyls, glutathione moieties, and combination thereof.
  • covalent adduct refers to the moiety that is transferred from the donor molecule to the acceptor in a group transfer reaction; such as sulfonate, phosphate, and glucuronic acid respectively for SULTs, kinases, and UGTs.
  • donor-product refers to the product of a group transfer reaction that is the fragment of the donor molecule that is generated when the covalent adduct is transferred to acceptor. Often it is a nucleotide (naturally occurring or synthetic) such as a PAP, UDP or ADP; or a non-nucleotide such as a s-adenosylhomocysteine , nicotinamide or a CoenzymeA.
  • the donor-product is detected by a general reaction including a first complex + donor-product — > second complex + displaced detectable tag.
  • tracer refers to a displaced detectable derivative or tag of a donor product that retains its structure sufficiently to bind to a specific antibody.
  • donor refers to a substrate for an enzyme catalyzing a group transfer reaction that carries the activated covalent adduct.
  • suitable donors include not only nucleotides, but also s-adenosyl methionine and acetyl-CoA, among others.
  • donor-X is another term for donor molecule in which X represents the covalent adduct.
  • acceptor refers to a substrate for an enzyme catalyzing a group transfer reaction to which the covalent adduct becomes covalently attached, wherein the substrate is a polypeptide, a protein, a nucleic acid, a carbohydrate, a lipid or a small molecule substrate such as a steroid or an amino acid.
  • acceptor-X refers to a reaction product in which X is the covalently bound covalent adduct; wherein the covalent adduct includes at least one of a phosphate, a sulfate, a carbohydrate, a naturally occurring amino acid, a synthetically derived amino acid, a methyl, an acetyl, or a glutathione moiety, and a combination thereof.
  • the covalent adduct is capable of altering either the function, the stability, or both the function and the stability of the acceptor substrate.
  • catalytically active enzyme refers to at least one of a sulfotransferase, a kinase, a UDP-glucuronosyltransferase, a methyl transferase, an acetyl transferase, a glutathione transferase, or a ADP-ribosyltransferase.
  • catalytic activity refers to a chemical catalytic activity, an enzymatic activity, or a combination thereof.
  • first complex refers to a complex having a macromolecule (i.e., an antibody or an inactivated enzyme) and a detectable tag.
  • second complex refers to a macromolecule and the donor product wherein the detectable tag is competitively displaced by the donor-product.
  • the term "observable” as used herein refers to detectable change in fluorescence, fluorescence intensity, fluorescence lifetime, fluorescence polarization, fluorescence resonance energy transfer (FRET) or chemiluminescence of the second complex or the displaced detectable tag and a combination thereof to obtain a measured observable.
  • the measured observable is compared with a characterized observable, wherein the characterized observable corresponds to the first complex.
  • detectable tag refers to a fluorescent or chemiluminescent tracer, which is conjugated to a donor product. Fluorescence is the preferred mode of detection for the invention.
  • a suitable detectable tag may be produced by conjugating for example, a chemiluminescent tag or a fluorophore tag, to the donor product molecule in such a way that it does not interfere significantly with antibody binding (i.e., most likely attached via the base portion of the nucleotide).
  • Fluorophores applicable to the methods of the present invention include but are not limited to fluorescein, rhodamine, BODIPY, Texas Red, Alexa Fluors and derivative thereof known in the art.
  • Rhodamine conjugates and other red conjugates may be synthesized and optimized as detectable tags because their higher wavelength emission is less subject to interference from autofluorescence than the green of fluorescein.
  • Chemiluminescent tags applicable to the invention include Lumigen TMA-6 and Lumigen PS-3 (Lumigen, Inc., Southfield, MI) which have adequate chemiluminescence quantum yield. These reagents possess an easily measured signal by virtue of an efficient chemiluminescent reaction with a predictable time course of light emission. Furthermore, chemiluminescent tags contribute little or no native background chemiluminescence. Also, measurement of light intensity is relatively simple, requiring only a photomultiplier or photodiode and the associated electronics to convert and record signals.
  • chemiluminescent signals can be generated in an immunoassay using enzyme fragment complementation methods, where the detectable tag would be the donor product conjugated to fragment A of a reporter enzyme, and its displacement from antibody by the donor product generated in the group transfer reaction would allow it to associate with fragment B of the enzyme resulting in formation of a catalytically active reporter enzyme capable of acting on a chemiluminescent substrate.
  • This method has been described using ⁇ -galactosidase as a reporter enzyme in U.S. patent no. 4,708,929.
  • immunoassay may refer to a number of assay methods wherein the product is detected by an antibody such as for example a homogenous assay, homogeneous fluorescence immunoassay, a homogeneous fluorescence intensity immunoassay, a homogeneous fluorescence lifetime immunoassay, a homogeneous fluorescence polarization immunoassay (FPIA), a homogeneous fluorescence resonance energy transfer (FRET) immunoassay or a homogenous chemiluminescent immunoassay, or a non-homogenous assay such as enzyme-linked immunoassay (ELISA) and a combination thereof.
  • a homogenous assay such as for example a homogenous assay, homogeneous fluorescence immunoassay, a homogeneous fluorescence intensity immunoassay, a homogeneous fluorescence lifetime immunoassay, a homogeneous fluorescence polarization immunoassay (FPIA), a homogeneous
  • Fluorescence polarization immunoassay refers to an immunoassay for detecting the products of group transfer reactions.
  • Fluorescence polarization FP is used to study molecular interactions by monitoring changes in the apparent size of fluorescently-labeled or inherently fluorescent molecules (Checovich, W. J., et al., Nature, 1995, 375:254-6; Owicki, J. C, J Biomol Screen, 2000, 5:297-306).
  • FP Fluorescence polarization
  • the tracer is bound to a much larger receptor, thereby increasing its effective molecular volume, its rotation is slowed sufficiently to emit light in the same plane in which it was excited.
  • the bound and free states of the fluorescent molecule each have an intrinsic polarization value, a high value for the bound state and a low value for the free state.
  • the measured polarization is a weighted average of the two values, thus providing a direct measure of the fraction of the tracer molecule that is bound.
  • Polarization values are expressed as millipolarization units (mP), with 50OmP being the maximum theoretical value.
  • figure 1 shows a schematic of a competitive FPIA for the SULT reaction product PAP in which the PAP produced from the SULT reaction competes with the tracer (fluorescently tagged PAP), for binding to anti-PAP antibody.
  • the starting polarization of the tracer is high because it is almost all bound to antibody, and it decreases as the reaction proceeds and the tracer is displaced from the antibody.
  • the amount of PAP produced in a SULT reaction can be quantified by using the following equation:
  • HTS high throughput screening
  • library refers to a plurality of chemical molecules (test compounds), a plurality of nucleic acids, a plurality of peptides, or a plurality of proteins, and a combination thereof.
  • screening is performed by a high- throughput screening technique, wherein the technique utilizes a multiwell plate or a microfluidic system.
  • binding molecule or “macromolecule” as used herein refers to an antibody or an inactivated enzyme.
  • the term "antibody” as used herein refers to a monoclonal, a polyclonal or recombinant antibody.
  • the antibody is produced against a donor-product of a group transfer reaction and is able to preferentially distinguish between a donor-product and a donor in the presence of a high donor concentration.
  • the antibody also exhibits specificity towards at least one of a phosphate portion of a nucleotide, (i.e., an ability to distinguish between a 5'- phosphate, a 5'-phosphosulfate, a 5 '-diphosphate and a 5' -triphosphate).
  • the antibody of the present invention differentiates with high stringency between PAP and PAPS.
  • the donor-product molecule for the SULT reaction, PAPS differs only by the addition of a sulfate group linked to the 5' terminal phosphate. This may seem like a relatively small structural difference, but the demonstrated ability of antibodies to discriminate between molecules that differ by a single phosphate - which is very similar in size and structure to a sulfate group - provides an important feature of the present invention. For example, an antibody may be raised against the ribosyl phosphate portion of the molecule. Furthermore, the FPIA- based SULT assay method of the present invention suitably requires an antibody that specifically binds the reaction product PAP in the presence of excess PAPS.
  • antibodies that specifically recognize ADP and not ATP may be generated in animals or by in vitro recombinant methods using ADP conjugated to a carrier protein in such a way that the phospho-ribosyl portion of the molecule is exposed, but the adenine portion is largely hidden from immunoreactivity.
  • a nonhydrolyzable analog of ADP such as one containing a methylene or sulfur group bridging the alpha and beta phosphates in order to prevent hydrolysis of the immobilized hapten by nucleotidases or phosphatases in the immunized animals.
  • antibodies that specifically recognize small molecules other than nucleotides, respectively s- adenosyl homocysteine and Coenzyme A are used.
  • inactivated enzyme refers to a binding molecule that may bind to the donor product.
  • ADP is the donor product and an inactivated nucleoside diphosphate (NDP) kinase may be the binding molecule.
  • NDP nucleoside diphosphate
  • a selective destruction of the catalytic activity with preservation of the binding properties by genetic engineering, allosteric inhibition, or other chemical means such as taking out cofactors, heme groups, etc. may enable enzymes, particularly multi-subunit enzymes to function as specific carriers for their substrates that are no longer able to chemically modify these substrates.
  • antibody-detectable tag pair refers to an anti-donor product antibody and detectable tag (fluorescent-donor product) molecule that allow detection of donor product produced in a group transfer reaction.
  • a suitable dissociation constant for binding of the antibody/detectable tag pair is 1x10 "5 M or lower, resulting in a minimal fluorescence polarization shift of 5OmP relative to the unbound detectable tag, and with minimal cross reactivity to the donor molecule at reaction concentrations.
  • the optimal antibody- detectable tag pair may be identified by testing a number of different antibodies generated using different sites of attachment to the donor molecule, different linkers to the carrier protein, and including some non-hydrolyzable analogs for interaction with a set of detectable tags generated by varying the chemistry of donor molecule attachment to a fluorophore.
  • the changes in fluorescence or chemiluminescence of the detectable tag upon interaction with an antibody that could have a fluorophore for example attached t it; may be used as a measure of their interaction.
  • linker refers to spacer arm structures that join the donor product to the carrier protein in the immunogen or to the fluorophore in the detectable tag.
  • linker molecules affects detectable tag characteristics in a number of important ways that impact both its antigenic and fluorescence properties. There is generally a balance that must be struck between separating the antigen from the fluorophore enough to allow unhindered interaction with antibody without creating too much freedom of motion for the fluorophore. The former result in lowered affinity antibody binding and in quenching of the fluorophore, whereas the latter reduces the polarization shift upon antibody binding, thereby reducing the dynamic range of the assay.
  • linker can be interchanged using relatively simple chemistry, thus the linker is varied in a number of ways in efforts to optimize the antigenic and fluorescence properties of the detectable tag.
  • approaches may be employed similar to those described for PAP conjugation of an antibody involving heterobifunctional linkers to first introduce spacer arms and/or aromatic substituents onto PAP, followed by reaction with reactive fluorescein derivatives. This approach greatly expands the range of possible linker structures.
  • the affinity and specificity of the antibody-detectable tag pair, and the resultant changes in detectable tag fluorescence properties ultimately define the overall performance of the assay, including sensitivity, dynamic range, and signal to noise ratios.
  • applicants have provided an iterative co-development strategy for these key assay reagents.
  • This co-development strategy provides that several different antibodies be tested to identify one that specifically binds the donor product in the presence of excess donor molecule.
  • the donor molecule may be conjugated to carrier protein (e.g., BSA or KLH) via attachment through different positions using linker molecules of varying lengths with reactive termini; e.g.
  • the same donor-linker reactive intermediates used for carrier protein conjugation may be used to synthesize fluorescein-PAP conjugates to test as detectable tags.
  • the resulting matrix of antibodies and detectable tags may be tested for binding using FP assays, and the pairs that exhibit high affinity binding and maximal FP shifts may be used as the basis for further optimization of tracer characteristics. Optimized antibody- detectable tag pairs may be tested for detection of donor molecule production in enzymatic reactions.
  • donor molecule antigens and detectable tags may be synthesized by conjugating the donor molecule to carrier protein and fluorescein, while retaining an overall structural bias that maximizes antibody recognition of the part of the molecule that differs from the donor so that crossreactivity with the donor is minimized.
  • this generally means attaching the linker to the base portion of the nucleotide so that the ribosyl-phosphate moiety is unaltered and fully exposed.
  • adenine nucleotide donor product it is known in the art that conjugation to adenine will minimize antibody cross reactivity that could occur via binding to the adenine moiety (Goujon, L., et al., J. Immunol Methods, 1998, 218: 19-30; Horton, J.K., et al, J Immunol Methods, 1992, 155:31-40; Bre resortt, R., et al., Biochim Biophys Acta, 1981, 652:16-28).
  • the site of hapten conjugation also can affect the affinity of the resulting antibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8).
  • some larger donor products such as Coenzyme A, it may be desirable to use only a portion of the molecule as antigen so that crossreactivity with the structural elements shared with the donor are minimized.
  • antibodies against a range of donor product antigens are suitably generated in rabbits. It is difficult to predict how the structure of the antigen will affect the affinity and selectivity of the resulting antibodies, but the carrier protein and mode of conjugation both can have a profound effect (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8; Signorini, N., et al., Chem Res Toxicol, 1998, 11 :1169-75; Oda, M. and Azuma, T., MoI Immunol, 2000, 37:1111-22). Accordingly, antibodies may be generated using conjugation to two different proteins via different sites on the donor product and using different length linker regions.
  • the injection of animals and collection of serum may be performed according to the following protocol.
  • Three rabbits may be immunized with each of the antigens that may be developed.
  • the yields of antiserum from a single rabbit are suitable for many thousand to millions of FPIA assays depending on the titer, affinity, and the multivalent nature of polyclonal-antigen interactions resulting in very high affinity binding.
  • polyclonals are frequently used for FPIAs (Nasir, M. S. and Jolley, M. E., Comb Chem High Throughput Screen, 1999, 2:177-90).
  • monoclonal antibodies which may be produced according to the methods provided in previously published protocols (Harlow, E. L., D., 1999), which are fully incorporated herein by reference.
  • mice are widely used for generation of monoclonal antibodies
  • rabbit hybridoma technology has also recently become available. Rabbits are believed to be more effective than mice for generation of high selectivity antibodies against small haptens, thus rabbit monoclonal antibodies may prove to be a better approach for generating the high selectivity and affinity desired for this application.
  • recombinant single chain antibodies may be generated using the in vitro combinatorial evolution and display methods previously published (Schaffitzel, C, et al., J Immunol Methods, 1999, 231 :119-35; Breitling, F., Dubel, S., 1999). Examples of the preparation of recombinant antibodies are described in U.S. Patent Nos.: 5,693,780; 5,658,570; 5,876,961 (fully incorporated by reference herein).
  • bovine serum albumin BSA
  • KLH antigens generally elicit a stronger immune response in mammals, but also tend to be less soluble than BSA conjugates (Harlow, E. L., D., 1999).
  • Donor products with an adenine ring such as ADP and PAP may be attached to both carrier proteins via the C2, C8 and N6 amino group of adenine.
  • Donor products with a uridine ring can be conjugated via the C5 or C6 position of uridine.
  • Either type of nucleotide may be conjugated through the ribose 2' and 3' hydorxyls. Conjugation Methods
  • hapten conjugation chemistries used by the invention are well described single or two step synthetic approaches that have been used extensively for conjugating nucleotides for generation of immunogens or for immobilizing them on solid supports (Brodelius, P., et al., Eur J Biochem, 1974, 47:81-9; Lindberg, M. and Mosbach, K., Eur J Biochem, 1975, 53:481-6; Camaioni, E., et al., J Med Chem, 1998, 41 :183-90). Most of the starting materials are commercially available, or can be obtained through custom synthesis, and it is understood that the reactions may proceed in good yield. These conjugation methods are described in the Examples in further detail.
  • the present invention may encompass but may not be limited by the hapten conjugation chemistries described herein. It is also envisioned that about 8-10 different antigens may be synthesized in all. Some antigens will have very short linkers such as C2 or C3 that will sterically minimize the accessibility of the base and others will have longer six carbon linkers that will allow more flexible presentation of the antigen.
  • an FPIA detectable tag molecule can be divided into three different structural components: the antigen, the fluorophore, and the linker used to join them; an additional key structural variable is the site of attachment of the linker to the antigen.
  • suitable antigens, fluorophores and linkers are not limited to what is specifically described herein.
  • the same donor product-linker molecules that are used for conjugation to carrier proteins may be used for conjugation to several reactive fluorophore derivatives.
  • the use of a range of reactive fluorophores may introduce additional structural diversity in the linker region.
  • fluorescein succinimidyl esters are available with different length spacer arms, and DTAF (4,6- Dichlorotriazinylamino fluorescein) contains a bulky planar substituent adjacent to the site of attachment.
  • DTAF 6,6- Dichlorotriazinylamino fluorescein
  • various donor product-linker molecules with reactive termini such as amino groups may be reacted with amine-reactive fluorophore derivatives and the resulting donor product-fluorophore conjugates may be tested for antibody binding affinity and changes in fluorescence polarization.
  • TLC thin layer chromatography
  • the difference in polarization of the probe in the free and bound states defines the total "spread" or dynamic range for the assay.
  • a change of less than 50 mP may be sufficient for semi-quantitative detection of enzyme activity, but a change of 100 mP or greater may provide both much greater flexibility in designing the assay format, and more quantitative kinetic information. (It should be noted that improvements in instrumentation may soon make it possible to accurately detect very small changes in polarization.)
  • polarization is proportional to molecular volume, and the change in effective volume upon binding of an antibody (150 kDa) to a small molecular weight fluorophore may be expected to cause an increase of at least 300 mP.
  • the initial screen involves looking for the following desirable properties: low polarization in the absence of antibody ( ⁇ 100mP); high affinity binding to the anti-donor product antibodies (K d ⁇ l ⁇ M); maximal difference in polarization between the bound and free states ( ⁇ mP >100mP); minimal fluorescence quenching upon binding antibody; selectivity for donor product (i.e., competitive displacement by donor product and not by donor molecule; rapid association/dissociation kinetics; and lack of interaction with other assay components.
  • the methods of the invention can be used as a measure of enzymatic activity in group transfer reactions.
  • the methods are of particular importance in the pharmaceutical industry since they enable the analysis of group transfer enzymes in high throughput screening laboratories, e.g. for identification of drugs that can act as enzymatic modulators, especially inhibitors, and for determining how potential drug molecules are metabolized.
  • kits of the present invention allow for the screening of potential drug molecules with SULTs, UGTs, methyltransferases and acetyltransferases in an HTS format.
  • the kits of the present invention would greatly simplify their testing in a matrix fashion.
  • the general methods of the invention may be applied to a variety of different group transfer-related enzymatic processes, such as steroid hormone biosynthesis and function, xenobiotic metabolism, enzyme receptor regulation, and signal transduction in an effort to contribute to an integrated drug discovery approach discussed below.
  • Sulfation is a ubiquitous cellular conjugative reaction used to modulate the function of endogenous biomolecules including proteins, carbohydrates, and small molecular weight signaling molecules like catecholamines and steroid hormones (Strott, C.
  • Sulfotransferase enzymes catalyze the conjugation of sulfonate groups onto a variety of xenobiotic and endogenous substrates primarily at hydroxyl groups to form a sulfate, but also at some aromatic amines resulting in sulfamate formation.
  • PAPS 3'-phosphoadenosine 5'-phosphosulphate
  • PAP 3',5'- bisphosphate adenine ribonucleotide
  • SULTs 3',5'- bisphosphate adenine ribonucleotide
  • the membrane bound enzymes are located in the golgi apparatus and sulfonate large endogenous molecules such as heparan, glycosaminoglycans, and protein tyrosines.
  • the cytosolic sulfotransferases, or SULTs metabolize small molecular weight xenobiotics and hormones.
  • Sulfation is reversible and can change the activity of signaling molecules, often by altering their affinity for receptor proteins, thus it serves as an on-off switch for receptor ligands, much as phosphorylation serves that role for proteins.
  • the role of sulfation in xenobiotic metabolism is intertwined with its involvement in the regulation of hormonal signaling and cell homeostasis. For instance, sulfation regulates the activity of endogenous ligands for specific neurotransmitter receptors, nuclear receptors, and protein kinases that are drug targets for depression, breast cancer, and cardiovascular disease (Plassart-Schiess, E. and Baulieu, E.
  • cytosolic SULT isoforms which differ in their tissue distribution and specificity.
  • the nomenclature used is based on homology at the amino acid level: SULTs within the same family, indicated by a number, share at least 45% amino acid identity, and those within the same subfamily, designated by a letter, share at least 60% identity.
  • the SULTs can be differentiated to some degree based on substrate specificity, though their substrate profiles overlap extensively, even between enzymes in the different families.
  • the assays of the invention may be used in an HTS format to provide for example, SULT metabolism information such as whether the compound of interest interacts with one or more SULT isoforms, whether the compound is a substrate or inhibitor, and the kinetic parameters (ICs 0 , K m , V max ) for the interaction.
  • the HTS assay encompassed by the present invention provides all of this information by screening in two different modes: a) direct measurement of test compound turnover; and b) measurement of compound inhibition of probe substrate sulfation. In the direct turnover mode, compounds could be screened vs. a panel of SULT isoforms to profile compound sulfation by individual SULT isozymes.
  • HTS assays of the invention enable a rational, integrated approach to drug development for therapeutic areas where for example, sulfation is a component of the relevant signal transduction biology.
  • Some specific areas where the HTS methods of the invention may be used include for example steroid hormone based therapies.
  • Sulfation in accordance with the present invention, encompasses involvement in estrogen level regulation in mammary tumors, as well as androgen levels in prostate tumors.
  • Cortisol sulfation though not well understood, inactivates the hormone for binding to the glucocorticoid receptor.
  • the methods encompassed by the present invention may suitably accelerate these efforts by allowing facile screening of endogenous and synthetic neurosteroids for sulfoconjugation, offering insight into the fundamental biology as well as providing a tool for lead molecule identification and optimization.
  • the need for better molecular tools is accentuated by the fact that there is already a sizeable over the counter market for DHEA as an "anti-aging" dietary supplement purported to alleviate age related senility and memory loss (Salek, F. S., et al., J Clin Pharmacol, 2002, 42).
  • the methods of the present invention may suitably identify drug targets with respect to cholesterol sulfate in the regulation of cholesterol efflux, platelet aggregation and skin development in treatments for cardiovascular disease and perhaps some forms of skin cancer.
  • a sulfotransferase - most likely SULT2Blb - could become the drug target, and molecules that selectively inhibit this isoform may need to be identified.
  • the availability of a full panel of the human SULTs and a robust HTS assay method of the present invention may be valuable to such an effort.
  • Uridine diphosphate-glucuronic acid (UDPGA) + aglycone -> UDP + glucuronide [000124] A broad spectrum of drugs are eliminated or activated by glucuronidation including non-steroidal anti-inflammatories, opioids, antihistamines, antipsychotics and antidepressants (Meech, R. and Mackenzie, P. I., Clin Exp Pharmacol Physiol, 1997, 24:907-15; Radominska-Pandya, A., et al., Drug Metab Rev, 1999, 31:817-99).
  • UGT families e.g., UGTl and UGT2
  • UGTl and UGT2 have been identified in humans; although the members of these families are less than 50% identical in primary amino acid sequence, they exhibit significant overlap in substrate specificity.
  • UGT2B4, 2B7, 2B10, 2Bl 1 and 2Bl 5 are expressed in the liver.
  • UGTs are known to have deleterious effects, including hyperbilirubinaemia which occurs with a frequency of 5-12% (Weber, W., 1997) and can lead to neurotoxicity and in severe cases, death.
  • Other drug metabolizing enzymes such as P450s
  • interindividual differences in UGT expression levels have been observed and linked to differences in drug responses (Iyer, L., et al., J Clin Invest, 1998, 101:847-54).
  • low expression of UGTlAl as in patients with Gilbert's syndrome, has been associated with the toxicity of Irinotecan, a promising anticancer agent (Wasserman, E., et al., Ann Oncol, 1997, 8:1049-51).
  • UGT IAl is clearly the predominant isoform involved in glucuronidation of the tetrapyrrole, bilirubin, resulting in its excretion. Beyond this, it is difficult to make generalizations regarding specificity because of the lack of systematic studies with most of the recently identified isoforms. Numerous endogenous steroids have been identified as aglycones for most of the hepatic isoforms include including IAl, 1 A3, 1 A4, 2B4, 2B7, and 2B15. Lipids and bile acids serve as substrates for 2B4 and 2B7, and recently retinoids have been identified as substrates for some isoforms from both families.
  • xenobiotic aglycones The structural diversity of known xenobiotic aglycones is very broad; it includes many drugs and drug like molecules including tertiary amines such as imipramine, non-steroidal antiinflammatories (NSAIDs) such as acetominophen and naproxen, opioids such as morphine and codeine, and carboxylic acid containing drugs such as clofibric acid.
  • NSAIDs non-steroidal antiinflammatories
  • opioids such as morphine and codeine
  • carboxylic acid containing drugs such as clofibric acid.
  • the immunoassay i.e., FPIA-based donor product assay
  • UGTs will be used in a manner very similar to that described for SULTs for determining whether potential drug candidates interact with any of the known UGT iso forms.
  • FPIA-based donor product assay FPIA-based donor product assay
  • the kinetic parameters IC 5O , K m , V max ) for the interaction between the compound of interest and enzymatic isoform.
  • the identification of the UGT responsible for the metabolism of a drug will aid in judicious selection of the in vitro assays or animal models used for preclinical assessment of possible drug-drug interactions and toxicology testing, thereby reducing inappropriate or unnecessary use of animals for experiments.
  • metabolism data can be used as a component of rational drug design.
  • a better understanding of the structure- activity relationships that define substrate specificity for the various UGT isozymes would provide a basis for structural modifications of primary compounds to change their metabolism profile.
  • the testing of glucuronidated compounds can lead to the discovery of valuable prodrugs that are inactive until metabolized in the body into an active form.
  • Protein kinases catalyze the transfer of the terminal phosphate group from ATP or GTP to serine, threonine or tyrosine residues of acceptor proteins:
  • kinases encoded in the human genome; elucidating their role in disease and identifying selective inhibitors is a major pharma initiative.
  • Kinase malfunction has been linked to all of the most important therapeutic areas, including cancer, cardiovascular diseases, inflammation, neurodegenerative diseases, and metabolic disorders.
  • clinical validation of kinases as drug targets has recently been shown in the cases of Herceptin and Gleevec, which inhibit aberrant tyrosine kinases that contribute to breast cancer and leukemia, respectively.
  • High throughput screening (HTS) the parallel testing of many thousands of compounds for interaction with a drug target - has become the dominant mode of drug discovery.
  • HTS assay reagents The total market for HTS assay reagents in 2002 was $474 million, and approximately 20% of screening was done on protein kinases.
  • pharma efforts to incorporate kinases into HTS programs are hampered by shortcomings with the assay methods.
  • the most commonly used HTS kinase assays rely on fluorescence-based immunodetection of a phosphorylated peptide reaction product, which varies with the substrate specificity of individual kinases. Time consuming reagent development is thus required for each kinase, or group of related kinases, and comparison of results between assays is problematic.
  • the invention provides for a FPIA for detection of adenosine diphosphate (ADP), a product of all kinase reactions.
  • ADP adenosine diphosphate
  • This assay will accelerate efforts to define kinase substrate specificity and to identify novel inhibitors by providing a universal catalytic assay that can be used with any kinase and any acceptor substrate.
  • Protein Kinases are a large, diverse family with a key role in signal transduction. Protein kinases, which catalyze the transfer of the terminal phosphate group from ATP or GTP to serine, threonine or tyrosine residues of acceptor proteins, are one of the largest protein families in the human genome. In the broadest senses, they can be divided into serine/threonine or tyrosine kinases and soluble enzymes or transmembrane receptors. In the most recent and comprehensive genomic analysis, 428 human kinases were identified that comprise eight different homology groups, which also reflect differences in substrate specificity, structure/localization and/or mode of regulation (Hanks, S.
  • Tyrosine Kinase group which includes both transmembrane growth factor receptors such as EGFR and PDGFR and soluble enzymes such as the Src kinases, 61 members of the cyclic nucleotide dependent group, ser/thr kinases which includes the lipid dependent kinases - the PKC iso forms, and 45 members of the "STE" group, which includes the components of the mitogenic MAP kinase signaling pathway.
  • kinases are ubiquitous regulators of intracellular signal transduction pathways, and as such have come under intense focus by pharmaceutical companies searching for more selective therapies for a broad range of diseases and disorders; they are second only to G-protein coupled receptors in terms of pharma prioritization (Cohen, P., Nat Rev Drug Discov, 2002, 1 :309- 15).
  • Intracellular targets for phosphorylation include other kinases, transcription factors, structural proteins such as actin and tubulin, enzymes involved in DNA replication and transcription, and protein translation, and metabolic enzymes (Cohen, P., Trends Biochem Sci, 2000, 25:596-601).
  • Phosphorylation can cause changes in protein catalytic activity, specificity, stability, localization and association with other biomolecules. Simultaneous phosphorylation at multiple sites on a protein, with different functional consequences, is common and central to the integration of signaling pathways. Diversity of Phosphorylation Sites.
  • Each kinase may phosphorylate one or more target proteins, sometimes at multiple sites, as well as autophosphorylate within one or more regulatory domains that control catalytic activity or interaction with other biomolecules.
  • Defining the functional consequences of cellular phosphorylation profiles for normal and disease states is a major proteomics initiative.
  • Kinases recognize specific linear sequences of their target proteins that often occur at beta bends. In general, amino acids that flank the phosphorylated residue for three to five residues on either side define a phosphorylation site.
  • the PhosphoBase database which compiles known kinase phosphorylation sites, contains entries for 133 human kinases, less than a third of the total kinases. Moreover, most, if not all of these specificity profiles are incomplete, as they only show one or two peptides that have been identified as substrates for each kinase. Though there is significant overlap in substrate specificity among related kinases, there is no consensus sequence that is phosphorylated by a large number of kinases. This situation complicates the incorporation of diverse or novel kinases into HTS assays that rely on detection of specific phosphorylated products.
  • RTKs Growth factor receptor tyrosine kinases
  • RTKs are membrane- spanning proteins that transduce peptide growth factor signals from outside the cell to intracellular pathways that lead to activation of progrowth and anti-apoptotic genes.
  • the majority of the fifty-eight RTKs in humans are dominant oncogenes, meaning that aberrant activation or overexpression causes a malignant cell phenotype.
  • tyrosine kinases are being aggressively pursued as anticancer drug targets and both small molecule and monoclonal antibody inhibitors - Gleevec and Herceptin, respectively - have been clinically approved.
  • Downstream signaling from growth factor receptors occurs through multiple pathways involving both ser/thr and tyrosine kinases.
  • One of the dominant kinases is the mitogen activated protein kinase (MAPK) pathway, which includes Raf and MEK kinases; inhibitors of all of these kinases are currently being tested in clinical trials (Table 1) (Dancey, J. and Sausville, E. A., Nat Rev Drug Discov, 2003, 2:296-313).
  • Bolded text refer to approved drugs.
  • Soluble tyrosine kinases especially the 11 oncogenes that comprise the Src family, also transduce mitogenic signals initiated by RTKs and are being targeted by pharma (Warmuth, M., et al., Curr Pharm Des, 2003, 9:2043-59). Following mitogenic signals through RTKs that initiate entry into the Gl phase, progression through the cell cycle is regulated by sequential activation of phase-specific kinases in association with cyclin proteins.
  • the cyclin dependent kinases represent yet another important group of kinases that pharma is pursuing in the hopes of inhibiting malignant cell proliferation (Table 1) (Elsayed, Y. A. and Sausville, E. A., Oncologist, 2001, 6:517-37).
  • the FPIA-based donor product assay will be used to screen drug libraries for inhibitors or activators of protein kinases. It will also be useful for screening peptides or proteins as acceptor substrates for kinases. In these applications, it will have the significant advantages over other methods such as the universal nature of the assay, simplified homogenous assay, no radioactivity, and the ability to quantify enzyme turnover. Universal Assay Method
  • This assay is a single addition, mix and read format. This is an important factor driving decisions on assay selection in an automated high throughput environment (High Tech Business Decisions, M., CA, Commisioned Market Analysis, 2002). In addition, if antibodies with suitable binding kinetics are isolated, it allows a continuous assay format that provides more detailed kinetic information than a stop-time assay. Fluorescence detection
  • the FPIA format eliminates radiation handling, disposal and costs. It should be noted that over the last few years FP has become one of the key
  • FP is a standard mode on several commercial HTS plate readers.
  • One embodiment of this application involves detecting the "donor product" of the UGT reaction using a competitive fluorescence polarization immunoassay where the antibody- bound tracer has a high polarization value which decreases when it is displaced by an analyte, such as UDP (as shown in Figure 2).
  • the main reagent required for this assay is the production of an antibody that binds UDP with high selectivity and has negligible binding to the donor, UDP-glucuronic acid (UDPGA).
  • UDPGA UDP-glucuronic acid
  • This highly selective antibody is used in combination with fluorescent UTP compounds (as substitutes for UDP tracers) to establish the assay.
  • the polyclonal antibody produced against UDP required covalent binding of the nucleotide hapten to a carrier protein.
  • UTP was used as the hapten only because reactive derivatives of the triphosphate, but not the diphosphate, were readily available that could be used for conjugation. It was reasoned that the majority of the triphosphate may be hydro lyzed to di- and monophosphate in animals.
  • Several different chemistries for linking the UTP to carrier protein were investigated, because the nature of the linkage can have a profound affect on the resulting antibody specificity and affinity for antigen. Care was taken so that the linker molecule was attached to the uridine ring rather than the ribose or phosphate, thus maximizing the immunoreactivity with the portion of the UDP molecule that may differentiate it from the donor, UDPGA.
  • One immunogen that proved particularly useful was 5-aminoallyl UTP (Sigma) conjugated to Keyhole Limpet Hemocyanin using glutaraldehyde.
  • the structure of the allyl linker is an important component of the tracer because it reduces the flexibility of the linker region relative to single bonds, preventing rotation of the fluor and resultant lower polarization values for the bound tracer.
  • Commercially available UTP tracers with alkynyl linkers (Molecular Probes, Corvallis, OR) have also worked well for the same reason. Applicants note that UTP was used for tracer synthesis for reasons of convenience because the aminoallyl derivative was commercially available. Tracers based on UDP have also been synthesized and have been shown to work equally well for the method of the invention, as would be expected. Selectivity of the antibody for UDP.
  • Figure 5 shows titrations of antibody- tracer complex with various uridine nucleotides using polyclonal antibody raised against a mixture of UTP and UDP conjugated to KLH (UGT-I).
  • the antibody and 2 nM of a commercially available Alexa 488-5-aminoalkynyl-UTP molecule (Molecular Probes) were added to wells of a black multiwell plate (Thermo Labsystems Pt#7605) containing the indicated amounts of undine nucleotides in 50 raM KPO4pH 7.5, 150 mM NaCl, 0.1 mg/ml BGG .
  • Fluorescence polarization was read in a Tecan Ultra plate reader after several hours of equilibration at room temperature using a Ex 485 /Em 535 filter set.
  • UTP and UDP compete off the tracer with similar effectiveness, with half maximal values (IC 50 ) of 5 and 15 ⁇ M, respectively.
  • the donor molecule UDP-glucuronic acid (UDPGA) competes much less effectively, with an IC 50 of approximately 2mM, indicating that this antibody is greater than 10Ox more selective for the reaction product UDP than the donor molecule, UDPGA using this displacement assay.
  • UDPGA UDP-glucuronic acid
  • UGT assays were performed in 35 ⁇ l volumes with varying amounts of UGT 2B7 BaculosomesTM (Invitrogen), 100 ⁇ M hyodeoxycholic acid (HDCA), and 70 ⁇ M UDPGA in the present of the anti UDP/UTP Ab/tracer complex.
  • the UTP tracer was synthesized in-house with AlexaFluorTM 488 (Molecular Probes).
  • the standard assay buffer consisted of 50 mM KPO 4 (pH 7.5), 150 mM NaCl, 5 mM MgCl 2 , and 1% DMSO (v/v). Incubations were at 37 0 C.
  • ⁇ mP mP (reaction with acceptor) - mP (reaction without acceptor).
  • FIG. 9 shows that the UGT assay can be used to detect glucuronidation of diverse acceptor substrates by different UGT isoforms, clearly illustrating the universal nature of the method.
  • UGT enzyme sources and acceptor substrates were tested in the UGT assay.
  • the enzyme protein concentration and the acceptor substrate @100 uM) for each of the reactions are as follows: 2B7 SupersomesTM (BD Biosciences)(50ug/ml, HDCA), HEK293 2B7 microsomes (kind gift from Dr.
  • the tracers are conjugates of 5-aminoallyl UTP to two different amine reactive Alexa Fluors, one with an excitation peak of 546 and the other with an excitation of 568. Both of these tracers are displaced from antibody (anti-UDP rabbit polyclonal Ab or UGT-I) in a similar manner by UDP. IC 50 values of 7 and 9 ⁇ M were observed for the Alexa 546 and Alexa 568 tracer, respectively. However, displacement by UDPGA differs significantly for the two tracers, with IC 5 o values of 1.2 and 6.4mM observed for the Alexa 546 and Alexa 568 tracer, respectively. Thus, the signal to background of the UGT assay with a given antibody can be enhanced by identifying tracers that are displaced by UDP more effectively than UDPGA. Signal stabilization and reaction quenching.
  • UGT enzymes are available only as crude membrane preparations that contain many other enzymes besides the desired UGT. Some of these contaminating enzymes can catalyze hydrolysis of the UDP molecule to UMP or Undine, resulting in an unstable signal for the assay. To prevent this, agents such as sodium vanadate and EGTA are added to stabilize the assay signal by preventing breakdown of the UDP formed during the UGT reaction.
  • Sodium vanadate at 3OmM prevents UDP breakdown and in addition very effectively inhibits UGT activity, so it is added after the UGT reaction is complete to quench the reaction and and stabilize the assay signal.
  • the use of such a quenching reagent provides greater flexibility in experimental protocols because HTS assay plates may be read at any time after the quench reagent is added.
  • the method does not require separation of reactants from products, and is a significant improvement over other related assays because it uses fluorescence detection rather than colorimetric, making it more sensitive and more desirable for pharma HTS platforms, which have become largely reliant on fluorescence based detection.
  • it is a continuous assay method, thus can provide real time kinetic data on UGT enzyme turnover.
  • the antibody-antigen binding reaction is less susceptible to interference from test samples than a conventional coupled enzyme reaction that has been used in the past for donor product detection (Mulder, GJ. and A. B. D. Van Doom, Biochem J., 1975, 151 : p. 131-40).
  • the novel assay method will enable screening of diverse compounds for metabolism by a panel of isolated UGT isozymes, which will greatly enhance preclinical metabolism studies, and potentially reduce the clinical attrition rate.
  • Glucuronic acid is one type of carbohydrate molecule that is activated by UDP for enzymatic transfer to other biomolecules; there are many other types of group transfer reactions that use UDP-sugar as an activated donor. These include protein glycosyltransferases that modulate the cellular localization and/or secretion of proteins via their carbohydrate modifications and biosynthetic enzymes that incorporate sugar monomers into polymers such as glycogen and bacterial peptidoglycan. Many of these enzymes are potential targets for therapeutic intervention, both in humans or for antimicrobials.
  • One class of microbial glycosyltransferases that are of interest from a drug discovery perspective include transferases involved in bacterial cell wall synthesis such as the UDP-galactofurnosyltransferase of Mycobacterium tuberculosis (Cren, S., Gurcha, S. S., Blake, A.J., Besra, G.S., Thomas, N.R. Org. Biomol. Chem. 2; 2418-2420 .
  • transferases involved in bacterial cell wall synthesis such as the UDP-galactofurnosyltransferase of Mycobacterium tuberculosis (Cren, S., Gurcha, S. S., Blake, A.J., Besra, G.S., Thomas, N.R. Org. Biomol. Chem. 2; 2418-2420 .
  • a mammalian glycosyltransferase that is of potential interest from therapeutic perspective is the O-linked protein glycosyltransferase (OGT), which transfers N-GIcNAc to serines and threonines of proteins, regulating their activity in a manner similar to phosporylation (Zachara, E.N., and Hart, G.W., Biochim. Biophys Acta 1673: 13-28.)
  • OGT O-linked protein glycosyltransferase
  • These and many other sugar transferases use UDP-activated donor molecules and thus can be detected using an anti-UDP antibody and UTP or UDP tracers described above, or similar antibody and tracer pairs.
  • glycosyltransferases that use TDP- or CDP-activated donors which may also be detected with similar reagents using the invention herein.
  • An antibody raised against UDP as described herein may have suitable cross reactivity for different purine diphosphates to be used for glycosytransferases that produce CDP or TDP.
  • different antibodies can be raised against these specific nucleotides.
  • 6-aminobutyl-ADP (Biolog Life Science Inst., Bremen, Germany) was conjugated to keyhole limpet hemocyanin with a suitable reagent such as glutaraldehyde and used to immunize rabbits.
  • a suitable reagent such as glutaraldehyde
  • Other carrier proteins such as BSA or ovalbumin and other conjugation chemistries may be used. Attachment to carrier protein via the adenine ring exposes the ribosyl-phosphate moiety, where selectivity is required, and minimizes exposure of the adenine ring.
  • the resulting antisera was tested for ADP binding affinity and selectivity in competitive fluorescence polarization binding assays.
  • Competitors included ADP (o), ATP (•), Adenosine ( ⁇ ), and AMP (A). These results are shown in Figure 12.
  • the half maximal point in the competition curve (IC 50 ) for ADP is 30OnM, whereas for ATP it is 15 ⁇ M; much higher amounts of AMP and adenosine are required to displace the tracer.
  • reaction mixture was shaken for 72 hours and products were purified via preparative thin layer chromatography (Whatman LK-6F) using 70:30 Ethanol/2 ml ammonium acetate. Fluorescent bands were scraped off and desorbed using 1 :1 Methanol/2 mM ammonium acetate, pH 5.5.
  • PKA Protein Kinase A
  • the final assay conditions were 50 mM HEPES buffer (pH 7.5), 1 mM EGTA, 0.1 mg/ml BGG, 50 uM kemptide substrate, 10 uM ATP, and 10 mM MgCl 2 , InM fluorescein-labeled tracer (N6-AB- ADP-5FAM), and rabbit anti-ADP proteinG-purified polyclonal antibody in a total volume of 30 uL.
  • the following amounts of PKA were used: ⁇ , 3.0 ng, ⁇ , 0.3 ng, A, 0.2 ng. Reactions lacking PKA were used as controls.
  • Figure 15 shows the assay results for PKA using FPIA.
  • the slope of the assay response curve over time increased with increasing amounts of PKA as would be expected for an enzymatic reaction.
  • the Alexa Fluor tracers were used in similar experiments with both polyclonal and monoclonal antibodies for detection of PKA activity.
  • the acceptor substrate used was a small peptide, but a peptide or an intact protein could also be used which is not possible with many of the other currently available kinase assay methods.
  • SULT 1 E 1 a SULT isoform
  • E. coli expression vector with a C-terminal 6x histidine tag was first subcloned into an E. coli expression vector with a C-terminal 6x histidine tag and the expressed protein was purified by affinity chromatography and characterized with respect to its physical and enzymatic properties.
  • the V max and estradiol K m values determined for the purified SULTlE compared favorably with published values for purified recombinant SULTlEl, which are 30-40nmol/min/mg and 5-15nM, respectively.
  • Small molecules like PAP must be conjugated to a carrier protein in order to be used as an immunogen.
  • an antigen density of 10-20 per carrier protein is optimal.
  • the two elements of our antigen synthesis strategy were a) synthesis and testing of several antigens because the site of attachment to nucleotide and linker structure can profoundly affect the properties of the resulting antibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8; Signorini, N., et al, Chem Res Toxicol, 1998, 11 : 1169-75; Oda, M.
  • An FPIA tracer molecule can be divided into three different structural components: the antigen, the fluor, and the linker used to join them; an additional key structural variable is the site of attachment of the linker to the antigen. Because identification of a tracer is largely empirical, applicants used a variety of linkers to join PAP and fluorescein via different sites on each molecule; in most cases the final linker region is a composite of the reactive fluorescein and PAP molecules used. [000168] The antibody strategy was to conjugate through the adenine moiety in order to generate antibodies that bind specifically to the ribosyl-phosphate group of PAP.
  • fluorescein was the preferred fluor used for conjugation. Though red-shifted fluors such as rhodamine are more desirable for HTS applications, development of FP tracers with fluorescein is usually more straightforward because it is less prone to non-specific binding effects and there are numerous activated derivatives available. As to the linker molecule, it affects tracer characteristics in a number of important ways that impact both its antigenic and fluorescence properties. There is generally a balance that must be struck between separating the antigen from the fluor enough to allow unhindered interaction with antibody without creating too much freedom of motion for the fluor.
  • N6-aminohexyl PAP was the only activated PAP molecule used for immunogen synthesis that was useful for tracer synthesis; the photoactivation reactions required to conjugate the two azido-PAP derivatives may be inefficient for joining two small molecules.
  • Figure 17 from left to right provides PAP molecules with amino-terminal linkers attached at the C8 and N6 position, amine- and carboxy-reactive fluorescein derivatives, and PAP molecules with carboxy- terminal linkers at the N6 and 2'-OH.
  • the fluorescent PAP conjugates were separated by TLC and tested for binding to anti-PAP antibodies.
  • sixteen reactions were run containing different combinations of PAP and fluorescein derivatives and each reaction yielded 1-4 fluorescent products that could be resolved by TLC. In all, more than 40 tracers have been purified and tested for binding to Ab.
  • N6-aminohexyl PAP Sigma
  • all of the reactive fluorescein derivatives are commercially available.
  • N6-carboxymethyl PAP 100 mg PAP was incubated with 0.3 g Iodoacetic acid in 1.2 mL aqueous, adjusted to pH 6.5 with LiOH. The reaction proceeded at 30°C for 5-7 days, periodically adjusting the pH to 6.5. The resulting 1-carboxymethyl-PAP product was precipitated with ethanol and reconstituted in distilled water and the pH was adjusted to 8.5 with LiOH. This reaction was heated at 9O 0 C for 1.5 hours to yield the N6- carboxymethyl PAP. This product was purified on a Dowexl-X2 (200-400 mesh) column equilibrated in 0.3 M LiCl, pH 2.75.
  • the expected succinate was column purified using Fast Flow Sepharose-Q resin (Pharmacia) equilibrated with 50 mL 250 mM NH 4 OAc, and eluted with a linear gradient of 500 mM to 750 mM NH 4 OAc (50 mL of each). Fractions containing the product were concentrated on a rotovap followed by lyophilization to furnish 8.4 mg or 67% yield as determined by absorbance measurement. Stock solution was stored at - 2O 0 C for future use.
  • Fluorescein labeled compounds - generally 3-5 produced in each reaction - were visualized using UV light, scraped from the TLC plates, and extracted from the silica gel using 1 : 1 MeOH/0.5 M NH4OAc. Individual fractions were shaken on a vortex for 1 hour wrapped in aluminum foil and centrifuged at 4000 RPM for 8 minutes. The supernatants were transferred to a separate labeled vial and the extraction process repeated. Combined supernatants were standardized to 50 nM solution for future use and frozen in amber micro fuge tubes at -2O 0 C.
  • the signal is proportional to the difference in the bound versus free tracer fractions, thus both the dynamic range and the sensitivity of the assay are dependent upon the affinity of the antibody for the tracer and the competing sample molecule.
  • a suitable dynamic range for an FPIA at least 70-80% of the tracer must be bound to antibody in the absence of competitor. This normally is achieved by using the tracer at a concentration 2-10 fold below the K d and the antibody at a concentration 2-3-fold over the Ka.
  • the useful concentration range of the assay then ranges from several-fold below the Ka concentration to about 20-fold over the K ⁇ j concentration.
  • the range of detection for the sample may be from about 2 nM to about 200 nM. If the Ka is ten fold less (1 nM), the sensitivity of the assay also improves about 10-fold.
  • Acceptable properties for anti-PAP antibody and tracer in terms of the assay parameters affected are defined as 1) dynamic range: low tracer polarization in the absence of Ab ( ⁇ 100mP) and maximal difference in polarization between the bound and free states ( ⁇ mP >100mP); 2) sensitivity: high affinity binding of tracer to the anti-PAP antibodies (Ka ⁇ l ⁇ M), displacement by free PAP with a similar IC 50 , and minimal fluorescence quenching caused by binding; 3) signal/noise: high Ab selectivity; i.e., competitive displacement of tracer by PAP and not by PAPS or other adenine nucleotides; also lack of tracer interaction with SULTlEl or other assay component; and 4) a continuous assay: rapid association/dissociation kinetics. Results of Ab-tracer interaction studies
  • Antibodies were serially diluted two-fold in 5OmM phosphate buffer (pH 7.4) containing 150 mM NaCl, 0.1 mg/mL BGG, and 1 nM tracer in a total volume of 100 uL in a 96-well plate or 12 ul in 384-well plates (results in the two plates are identical) and polarization values were read on the Tecan Ultra after one hour incubation at room temperature.
  • N6-PAP-6F8 tracer is represented by the open symbols: O(PAP), D (PAPS).
  • C8-PAP-F14 tracer is represented with the closed symbols: • (PAP), ⁇ (PAPS).
  • PAP or PAPS was serially diluted two-fold in black mutiwell plates containing 50 mM phosphate buffer (pH 7.4), 150 mM NaCl, 0.1 mg/mL BGG, 1 nM C8-PAP-F14, and 1.5 uL purified Ab 3642 in a total volume of 12 ⁇ l or 1 nM N6-PAP-F8 and 0.5 uL 3642 Ab in a 2OuL volume. Polarization values were read after one hour at room temperature.
  • the IC 50 for PAP with the C8-PAP-F14 tracer is 30OnM, low enough to allow use of these reagents for monitoring SULTlEl activity.
  • a much higher concentration of PAP (and PAPS) is required to compete off the tighter binding N6-PAP- F8 tracer. This may be because in this case the tracer has the same linker group as the immunogen used to generate antibody, and a population of antibody is recognizing the linker, making the tracer more difficult to displace with free PAP.
  • Figure 20 also shows that PAPS is less effective than PAP at displacing the tracers from Ab 3642.
  • FIG. 21 shows competition curves with two anti-PAP antibody/tracer combinations. Competitors were serially diluted two-fold in a black 384-well microtiter plate containing 50 mM phosphate buffer (pH 7.4), 150 mM NaCl, 0.1 mg/mL BGG, 1 nM Tracer C8-PAP-F14, and either 1.5 uL Ab 3642 (A) or 3 uL Ab 1781 (B) in a total volume of 12 uL.
  • Figure 22 illustrates the effect of PAPS concentration on detection of enzymatically generated PAP.
  • the assay mixture included 200 ng of SULTlEl -6xHis (G) or assay buffer ( ⁇ ) was added to wells containing 30 mM phosphate (pH 7.4), 7 mM DTT, 8 mM MgCl 2 , 75 mM NaCl, 0.5 mg/mL BGG, 150 nM estradiol, 1 nM C8-PAP-F14 tracer, 12.5 uL Ab 3642, and varying concentrations of PAPS in a total assay volume of lOO ⁇ L.
  • Figure 23 provides graphs of continuous FPIA-based detection of SULT activity with diverse substrates, acceptor substrates (200 nM) were added to wells containing 30 mM phosphate (pH 7.4), 7 mM DTT, 0.8 mM MgCl 2 , 75 mM NaCl, 0.5 mg/mL BGG, 2 uM PAPS, 200 ng SULTIEl-cHis, 1 nM C8-PAP-F14 Tracer, and 12.5 uL Ab 3642 in a total volume of 100 uL. Control reactions (top trace in each graph) lacked C-His-SULT1E1 and contained all other reaction components. The plate was incubated at room temp and polarization read at 1 minute intervals.
  • Figure 24 is a comparison of SULTlEl acceptor substrate profiles determined using the FPIA-based assay and the 35 S-PAPS radioassay. Rates of FPIA-based reactions were calculated from linear portions of curves shown in Figure 23. Acceptor substrates were used at 20OnM (FPIA) or 40OnM ( 35 S- radioassay).
  • Figure 25 is a graph showing inhibition of SULTlEl by 2,6 Dichloro-4-nitrophenol (DCNP) measured with the FPIA-based assay.
  • DCNP was serially diluted two-fold into wells in 46 uL of phosphate assay buffer (30 mM KPO 4 (pH 6.5), 0.5 mg/mL BGG, 15 mM DTT, 1.6 mM MgCl 2 , 4 ⁇ M PAPS), followed by 50 ⁇ l of a 2X Antibody/tracer mix (5 uL 3642 Ab/ 2 nM Tracer C8- PAP-F 14), and 200 ng of SULTlE in a total volume of lOO ⁇ l. The plate was incubated at room temp for 30 min, and read on the Tecan Ultra. ⁇ mP values were calculated by subtracting the SULTlE reactions from the no SULTlE controls. All values represent the mean of replicates.
  • an antibody suitably a monoclonal antibody with approximately 10-fold greater affinity and selectivity for PAP will be produced that will enable development of an assay with suitable dynamic range and signahnoise for commercial HTS applications.
  • Example 5 Assay Systems
  • the present assay system comprises an assay receptacle in which the assayed reaction is carried out, and a detector for detecting the results of that reaction.
  • the assay receptacle is selected from a test tube, a well in a multiwell plate, or other similar reaction vessel.
  • the various reagents are introduced into the receptacle and typically assayed in the receptacle using an appropriate detection system, described above such as a fluorescence polarization detector.
  • a flat surface such as glass or plastic could also be used and the reaction components spotted onto the surface in a defined array (such as a microarray).
  • the reaction receptacle comprises a fluidic channel, and preferably, a microfluidic channel.
  • microfluidic refers to a channel or other conduit that has at least one cross-sectional dimension in the range of from about 1 micron to about 500 micron.
  • microfluidic devices useful for practicing the methods described herein include, e.g., those described in e.g., U.S. Pat. Nos. 5,942,443, 5,779,868, and International Patent Application No. WO 98/46438, the disclosures of which are incorporated herein by reference.
  • an enzyme mediated coupling reaction between a first and second reactant may be carried out in channels of a microfluidic device.
  • a microfluidics platform it may be possible to mimic the compartmentalization of a eukaryotic cell. This method could then be used to monitor the activity of group transfer reactions catalyzed by enzymes in a more native environment, in the context of other proteins and with cellular components that may affect enzymatic activity. Therefore, data on the activity of enzymes that catalyze group transfer reactions and the consequences of their inhibition can be obtained in a setting that will more accurately reflect an in vivo environment.
  • these assay systems may be capable of screening test compounds that affect enzymatic reaction of interest.
  • devices used in accordance with the present invention are configured to operate in a high-throughput screening format, e.g., as described in U.S. Pat. No. 5,942,443.
  • test compounds instead of delivering potential test compounds to the reaction zone from a reservoir integrated into the body of the device, such test compounds are introduced into the reaction zone via an external sampling pipettor or capillary that is attached to the body of the device and fluidly coupled to the reaction zone.
  • Such pipettor systems are described in, e.g., U.S. Pat. No. 5,779,868 (fully incorporated by reference).
  • the sampling Pipettor is serially dipped into different sources of test compounds which are separately and serially brought into the reaction zone to ascertain their affect, if any, on the reaction of interest.
  • Movement of materials through the channels of these micro fluidic channel networks is typically carried out using any of a variety of known techniques, including electrokinetic material movement (e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by reference), pressure based flow, axial flow, gravity flow, or hybrids of any of these.
  • electrokinetic material movement e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by reference)
  • pressure based flow e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by reference)
  • pressure based flow e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by reference)
  • pressure based flow e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by reference)
  • pressure based flow e.g., as described in U.S. Pat. No., 5,858,195 (fully incorporated by
  • kits for detecting and quantifying a donor product of a group transfer reaction or a catalytic activity generating the donor-product of a group transfer reaction The general equation for the group transfer reaction includes a donor- X + acceptor -» donor-product + acceptor -X, wherein the donor-product is detected by the general detection reaction: first complex + donor-product — > second complex + displaced detectable tag.
  • the kit for an FPIA immunoassay may include a macromolecule (i.e., antibody or an inactivated enzyme) and a tracer (displaced) and optionally the specific group transfer enzyme of interest. It is noted that the macromolecule and the tracer may either be separate or incorporated into one solution vessel.
  • the kit may also include components such as, an activated donor, a detectable tag, acceptor substrates, inhibitors, buffers, cofactors, stabilizing agents, a set of instructions for using the kit, or packaging and any combination thereof.
  • the kit may be formatted for multiplex detection by using more than one antibody/detectable tag pair where the detectable tags can be differentiated on the basis of the observables they produce.
  • the immunoassay may be used to detect the donor product or the catalytic activity generating the donor product.
  • the kit may be used for screening a library for a molecule or a set of molecules, capable of contacting an enzyme, wherein the enzyme generates the donor-product in a group transfer reaction.
  • the library may include at least one of a plurality of chemical molecules, a plurality of nucleic acids, a plurality of peptides, or a plurality of proteins, and a combination thereof; wherein the screening is performed by a high-throughput screening technique using a multi-well plate or a micro fluidic system.
  • the macromolecule in the kit includes at least one of an antibody, a polypeptide, a protein, a nucleic acid molecule, an inactivated enzyme, and a combination thereof that is capable of contacting the donor-product with high affinity. It is further envisioned that the kit optionally include at least one of a sulfotransferase, a kinase or an UDP-glucuronosyl transferase, a methyl transferase, an acetyl transferase, a glutathione transferase, or an ADP-ribosyltransferase and combination thereof.
  • kits designed to be used for detecting donor product or the catalytic activity generating the donor product through other means such as a homogenous assay, a homogeneous fluorescence intensity immunoassay, a homogeneous fluorescence lifetime immunoassay, a homogeneous fluorescence resonance energy transfer (FRET) immunoassay or a homogenous chemiluminescent immunoassay, or a non-homogenous assay such as enzyme- linked immunoassay (ELISA).
  • a homogenous assay such as a homogenous fluorescence intensity immunoassay, a homogeneous fluorescence lifetime immunoassay, a homogeneous fluorescence resonance energy transfer (FRET) immunoassay or a homogenous chemiluminescent immunoassay, or a non-homogenous assay such as enzyme- linked immunoassay (ELISA).
  • FRET fluorescence resonance energy transfer
  • ELISA enzyme- linked immunoassay
  • the kit could be composed of an antibody and fluorescent detectable tag where the intensity and/or lifetime of the detectable tag is different when it is bound to antibody and than it is free in solution.
  • the difference in fluorescence; i.e., the assay signal could be enhanced by modification of the antibody such that its interaction with the detectable tag results in a further change in its fluorescence properties; i.e., quenching, enhancement, or a change in the lifetime.
  • a homogenous FRET immunoassay the interaction of a first fluor associated with the detectable tag with a second fluor that is attached to the antibody - either directly or via associated binding molecules such as biotin and streptavidin - could result in the excitation of the second fluor (or the reverse), thereby generating a fluorescence emission at a wavelength different from that of the detectable tag.
  • the second fluor could be a small organic molecule or a luminescent lanthanide probe. (It is noted that lanthanide emission is not fluorescence and is referred to as luminescence-based resonance energy transfer, or LRET).
  • the detectable tag could be the donor product bound to one fragment of an enzyme used for chemiluminescent detection such as ⁇ -galactosidase.
  • an enzyme used for chemiluminescent detection such as ⁇ -galactosidase.
  • the donor product-fragment-one complex is displaced from antibody by the donor product, it would then bind to fragment two of the enzyme, producing an intact, active enzyme that would be capable of producing a chemiluminescent signal with an appropriate substrate.
  • the assay would not be homogenous, and would require donor product or antibody be immobilized to the surface of multiwell plates. A secondary antibody conjugated to a detection enzyme would also be included in this format.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention porte sur des procédés de détection, quantification et criblage à haut débit de produits donneurs ainsi que sur les activités catalytiques produisant lesdits produits donneurs lors de réactions à transfert de groupe. L’invention concerne également des dosages immunologiques, des anticorps et des kits qui peuvent servir à la mise en œuvre de ces procédés.
EP05785285A 2005-05-26 2005-05-26 Procede d analyse destine aux reactions a transfert de groupe Withdrawn EP1869083A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2005/018645 WO2006127008A1 (fr) 2005-05-26 2005-05-26 Procede d’analyse destine aux reactions a transfert de groupe

Publications (1)

Publication Number Publication Date
EP1869083A1 true EP1869083A1 (fr) 2007-12-26

Family

ID=35170018

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05785285A Withdrawn EP1869083A1 (fr) 2005-05-26 2005-05-26 Procede d analyse destine aux reactions a transfert de groupe

Country Status (2)

Country Link
EP (1) EP1869083A1 (fr)
WO (1) WO2006127008A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004068115A2 (fr) * 2003-01-30 2004-08-12 Bellbrook Labs, Llc Procede relatif a des essais de reactions a transfert de groupe
US9163053B2 (en) 2007-05-18 2015-10-20 Fluidigm Corporation Nucleotide analogs
US7678894B2 (en) * 2007-05-18 2010-03-16 Helicos Biosciences Corporation Nucleotide analogs
WO2009078876A1 (fr) * 2007-12-18 2009-06-25 Bellbrook Labs, Llc Procédé de dosage pour des réactions de transfert de groupes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027421A2 (fr) * 2002-09-17 2004-04-01 Innova Biosciences Limited Dosage adp

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004027421A2 (fr) * 2002-09-17 2004-04-01 Innova Biosciences Limited Dosage adp

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MEYER T ET AL: "PRODUCTION OF ANTI-(ADP-RIBOSE) ANTIBODIES WITH THE AID OF A DINUCLEOTIDE-PYROPHOSPHATASE-RESISTANT HAPTEN AND THEIR APPLICATION FOR THE DETECTION OF MONO(ADP-RIBOSYL)ATED POLYPEPTIDES", EUROPEAN JOURNAL OF BIOCHEMISTRY, BERLIN, DE, vol. 155, no. 1, 17 February 1986 (1986-02-17), pages 157 - 165, XP008029164, ISSN: 0014-2956 *
MOHAMMED SARWAR NASIR ET AL: "Fluorescence Polarization: an analytical tool for immunoassay and drug discovery", COMBINATORIAL CHEMISTRY AND HIGH THROUGHPUT SCREENING, HILVERSUM, NL, vol. 2, no. 4, 1999, pages 177 - 190, XP001062258, ISSN: 1386-2073 *
See also references of WO2006127008A1 *
YUHASZ S C ET AL: "EPITOPIC DISCRIMINATION BY MONOCLONAL ANTIBODIES DIRECTED AGAINST THE SAME-ALKYLATED NUCLEOSIDE", JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS, ADENINE PRESS, NEW YORK, NY, US, vol. 7, no. 1, August 1989 (1989-08-01), pages 151 - 165, XP002043412, ISSN: 0739-1102 *

Also Published As

Publication number Publication date
WO2006127008A1 (fr) 2006-11-30

Similar Documents

Publication Publication Date Title
US20080234137A1 (en) Assay Method for Group Transfer Reactions
WO2009078876A1 (fr) Procédé de dosage pour des réactions de transfert de groupes
US8841136B2 (en) Levetiracetam immunoassays
EP2945942B1 (fr) Immunodosages de viroconazole
US7378505B2 (en) Assay method for group transfer reactions
JP7459018B2 (ja) バックグラウンドシグナルを補正するホモジニアスなイムノアッセイ
US20230417779A1 (en) Pregabalin Immunoassays
EP2956444B1 (fr) Dosages immunologiques du posaconazole
US8088897B2 (en) Assay method for group transfer reactions
US7847066B2 (en) Antibody that binds uridine diphosphate
EP1869083A1 (fr) Procede d analyse destine aux reactions a transfert de groupe
US20070178537A1 (en) Assays for detection of bioactive compounds that interact with heat shock protein 90
Fessenden Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method
Al-Shehri et al. Development and validation of an ELISA with high sensitivity for therapeutic monitoring of afatinib

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20071102

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20080408

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20110111