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Oct 15, 2006 (Vol. 26, No. 18)

Streamlining HTS for Challenging Targets

Screening Diverse Enzymes Using Homogeneous Nucleotide Detection

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    Figure 1a: Transcreener Kinase Assay principle.

    Adenine and guanine nucleotides are interconverted by diverse proteins of therapeutic interest, including kinases, phosphodiesterases, membrane transporters, DNA-modifying enzymes, and molecular chaperonins. Detection of nucleotide products, usually via radioassay or coupled enzymatic assays, has traditionally served as a convenient generic biochemical assay method for many of these protein families. However, translating these assays into an HTS format has not been practical because they require separation of reaction products or colorimetric endpoints.

    BellBrook Labs (www.bellbrooklabs.com) recently developed an HTS assay platform called Transcreener™ that enables fluorescent detection of nucleotides in a homogeneous or mix-and-read format.

    The assays rely on selective antibodies that are able to distinguish between nucleotides on the basis of a single phosphate group, and they have been formatted for robust ratiometric fluorescent readouts that minimize compound interference. Here we describe the use of Transcreener assays for ADP and AMP/GMP in detecting kinases, ATPases (chaperonins), and phosphodiesterases using either fluorescence polarization (FP) or time resolved fluoresence resonance energy transfer (TR-FRET).

    Adenine nucleotides provide energy to drive myriad cellular reactions, including the protein phosphorylation reactions central to signal transduction, and function directly as signaling molecules through binding to protein receptors inside and outside the cell. Hydrolysis of the phosphodiester bonds of ATP drives active transport through membrane channels; cell division, shape, and movement via organization of cytoskeleton proteins; and synthesis and repair of biomolecules including protein, nucleic acids, and lipids.

  • Metabolism and Signal Transduction

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    Figure B: Time resolved fluoresence resonance energy transfer

    Adenosine and its mono-, di- and tri-phosophate forms also act as allosteric effectors for many enzymes and serve as extracellular ligands for GPCRs and ion channels that regulate cell growth, differentiation, and neurotransmission. Cyclic adenine and guanine nucleotides serve as key second messengers for signal transduction for many GPCRs.

    Not surprisingly, many enzymes and receptors that utilize adenine nucleotides are being targeted for therapeutic intervention in diverse disease areas. Protein kinases are the best known example because of the clinical success of small molecule and antibody-based kinase inhibitors used for cancer treatment. In addition to the 518 human protein kinases, there are at least 150 non-protein kinases that phosphorylate lipids, carbohydrates, and nucleic acids. Lipid kinases have come under the most intense focus as therapeutic targets, with several companies currently developing PI3K inhibitors.

    Aside from the kinase superfamily, there is tremendous diversity in the types of adenine nucleotide–utilizing enzymes that are being targeted for therapeutic intervention both in humans and for anti-infectives, including molecular chaperonins, phosphodiesterases, and topoisomerases.

    To address the need for more flexible HTS assays for adenine nucleotide dependent enzymes, Bellbrook Labs developed fluorescence-based immunoassays for homogenous detection of ADP and AMP/GMP (Figure 1). The assays rely on antibodies that differentiate between nucleotides on the basis of small structural differences, such as a single phosphate group, with 100-fold or greater selectivity.

    For example, the monoclonal antibody developed for the Transcreener Kinase Assay binds ADP 140-fold more tightly than ATP, and polyclonal antibodies recently developed for AMP exhibit more than 1,000-fold selectivity over cAMP. Signal is generated when enzyme reaction products (AMP or ADP) displace a fluorescent tracer from antibody resulting in a change in its fluorescent properties. The assay method has been formatted for far-red FP and TR-FRET readouts (Figure 1), two commonly used detection modes that minimize interference from sample turbidity or fluorescence.

  • ADP Detection

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    Figure 2: Detection of diverse members of the kinase superfamily with the Tanscreener Kinase Assay.

    The advantages of the Transcreener platform for detection of diverse members of the kinase superfamily are illustrated in Figure 2. Most kinase assay methods rely on detection of a specific phosphorylated molecule that is produced by one or a small subset of enzymes, thus specific detection reagents and/or conditions are required for individual enzymes or subsets of enzymes.

    For example, antibody recognition of phosphoserine in peptides or proteins is dependent on the flanking amino acids, so many different antibodies would be required to detect the phosphorylated products of the hundreds of ser-thr kinases in humans. In contrast, the Transcreener Kinase Assay relies on detection of ADP, the invariant product of all kinase reactions, so the same reagents can be used for any kinase, regardless of the acceptor substrate. Figure 2 demonstrates the generic nature of the assay. Serine-threonine and tyrosine protein kinases are readily detected using either peptide or native protein acceptors as are lipid and carbohydrate kinases with their physiological substrates.

    The Transcreener ADP detection method provides flexibility for development and optimization of kinase screening parameters, providing opportunities for increased efficiencies in primary screening and lead optimization. For instance, a number of different peptide and/or protein substrates can be easily tested to identify the optimal acceptor for a target protein kinase.

    Profiling hits from a primary screen across a diverse panel of kinases to assess selectivity is also greatly simplified by the generic nature of the method. The Transcreener Kinase Assay does not have the ATP concentration limitations inherent in some other kinase assay methods, so ATP can be matched to the Km of the enzyme of interest.

    By providing a single method and output for all primary and secondary kinase screening functions the Transcreener platform has the potential to enable a more integrated and iterative approach to kinase drug discovery.

    Like kinases, ATPases catalyze a phosphoryl transfer reaction using ATP as a donor, resulting in the production of ADP. ATPases differ in that they transfer the gamma phosphate of ATP to water rather than a biomolecule. ADP detection with the Transcreener reagents provide a homogenous, fluorescent assay method for ATPases, shown in Figure 3 for the chaperonins HSC70 and HSP90.

    The method is perhaps even more enabling for screening ATPases than for kinases, because there are far fewer alternative assay methods. Colorimetric phosphate detection is the default method, and it is highly susceptible to interference from colored compounds common in screening libraries.

  • AMP Detection

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    Figure 3: Detection of HSC70 and HSP90 ATPase activity using the Transcreener Kinase Assay ADP detection reagents.

    Similar reagents were developed for the detection of AMP and GMP, primarly for screening cyclic nucleotide phosphodiesterases. As shown in Figure 4, one polyclonal antibody/tracer pair enabled detection of AMP and GMP with more than 1,000-fold selectivity over the corresponding cyclic nucleotides, making this single set of reagents suitable for screening any cyclic nucleotide phosphodiesterase. Initial validation experiments with human PDE 4A4 and 5, which are specific for cAMP and cGMP, respectively, have verified robust detection (Z´>0.5) of initial velocity (£10% substrate conversion) using starting concentrations of cyclic nucleotides as low as 100 nM.

    Moreover, the Transcreener AMP/GMP detection reagents exhibit more than 1,000-fold selectivity for AMP versus ATP, which should make them suitable for enzymes that convert ATP to AMP and pyrophosphate, such as 5´-nucleotide phosphodiesterases and diverse types of ligases.

  • TR-FRET Detection Module

    Click Image To Enlarge +
    Figure 4: Selectivity of Transcreener PDE assay detection reagents for adenine and guanine nucleotides.

    The first-generation Transcreener Assays use FP as a detection mode. To meet the diverse needs of the screening community, we recently formatted the kinase assay for TR-FRET as well (Figure 1). Both of these detection modes involve ratiometric measurements and so are much less subject to interference than direct fluorescence intensity measurements.

    The Transcreener Kinase TR-FRET Assay is a two-component detection module, making it simpler than other TR-FRET detection modules requiring three or more binding components. It incorporates Invitrogen’s (www.invitrogen.com) LanthaScreen™ lanthanide chelate-antennae complex technology for efficient energy capture.

    The FRET donor is a terbium chelate complex covalently attached to BellBrook’s ADP-specific monoclonal antibody. The acceptor is a fluorescein-ADP tracer. Displacement of tracer from antibody by ADP disrupts luminescence energy transfer from the terbium to the tracer, resulting in decreased fluorescein intensity (Figure 1b). The most advantageous feature of the TR-FRET format is that the assay signal is delayed relative to the prompt fluorescence from small organic fluors that are typically the source of assay interference.

    Both the FP and TR-FRET Transcreener Assay formats allow enzyme initial velocity measurements over physiological ranges of substrate concentrations, with Z´ values of greater than 0.5. The FP format utilizes a far red tracer (647-nm emission peak) to minimize interference from fluorescent compounds, and with TR-FRET, background fluorescence is minimized by time-gated detection. Adenine nucleotides are ubiquitous co-substrates for cellular reactions. The homogenous ADP and AMP/GMP Transcreener assays provide a generic screening method for diverse enzyme families encompassing hundreds of potential human, bacterial, and viral targets.



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