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Tutorials : Jun 15, 2005 ( )
Miniaturized Filtration Assay for Discovery
MultiScreenHTS 384-Well Filter Plate Increases Throughput of Filter-Based Assays
A major focus in drug discovery involves the identification of compounds that reduce or modulate biological processes by altering the activity of cellular receptors or signal transduction components such as kinases. Located in the cell membrane, the cytoplasm, or the cell nucleus, receptors are capable of highly specific binding to biological ligands. Upon the binding of a receptor to a ligand, signal transduction processes, including phosphorylation cascades mediated by kinases, regulate various biological processes required for cell growth and function.
For decades, heterogeneous filter-binding analyses have been employed, particularly under more demanding assay conditions (e.g., using unpurified tissue homogenates, cell membrane fragments, or cellular extracts), to study receptor binding and kinase activity.
Radiometric filter-binding assays are a proven methodology that directly measure either the extent of ligand binding to a wide range of receptors or, in the case of kinase screening, phosphorylation of target proteins and peptide sequences. Radiometric assays are less susceptible to artifacts generated by naturally fluorescent or quenching compounds (such as aromatic compounds). Filter-binding assays typically require minimal set-up and are fully automatable. The protocols are well documented in the peer reviewed literature.
Radiometric filter-binding assays have been successfully performed on 96-well platforms, but increasingly this format has fallen short of meeting the throughput and cost targets of secondary screening laboratories. This reality has fueled the development of higher throughput platforms that perform adequate library screening in a timely and cost-effective manner.
Millipore's (www.millipore.com) MultiScreenHTS 384-well filter plate (Figure 1) fulfills these needs in two different ways: by quadrupling the throughput of filter-based assays and by significantly reducing reagent consumption and costs. These benefits have been achieved without sacrificing assay sensitivity, robustness, or precision. The reduced filter-binding surface area and smaller assay volumes improve well-to-well reproducibility and reduce nonspecific binding. Furthermore, assays can be performed directly in the filter plate, which decreases the number of manipulations, reduces radioactive solid waste, and eliminates the need for a separate incubation plate.
Filter Plate-based Receptor Ligand Binding Assays
Many diseases are treated by the administration of drugs that regulate the activity of receptors and/or their downstream effector molecules. Therefore, a significant portion of drug discovery involves the systematic screening of large compound libraries for specific binding to various cellular receptors.
G-Protein Coupled Receptors (GPCRs), comprising the largest family of receptors in the human genome, are involved in a large number of disease states. One of the hallmarks of GPCRs is the fact that they span the cell membrane and have binding domains on both the cytoplasmic and extracellular surfaces.
This characteristic makes these receptors essentially impossible to purify from the cellular membrane in an active form. Techniques to characterize binding of GPCRs while they are present in the cellular membrane are highly desirable. For decades, filter-binding assays have satisfied the need to study GPCR binding in their natural co-existence with the cell membrane.1
Receptor ligand-binding assays typically performed in filter plates include saturation binding and displacement binding. Saturation binding involves generating serial dilutions of a ligand (some or all of which may be radiolabeled) in the presence or absence of excess unlabeled ligand.
Binding in the presence of excess, unlabeled ligand is used to determine assay nonspecific binding, which is then subtracted from total binding (absence of competing ligand) to calculate specific binding. Saturation-binding assays are ideal for the determination of the binding affinity that the receptor has for a ligand and for determining the specific activity of the receptor in a given receptor preparation.
Displacement binding is a method in which a constant amount of radiolabeled ligand is competitively displaced by a non-labeled, competitor ligand. It is the typical mode for screening large libraries of compound for their ability to displace a known (radiolabeled) ligand.
Titration of a nonlabeled ligand is used to estimate its relative binding affinity for the receptor (IC50, Ki). Alternatively, this technique can be used to screen large libraries of compounds by their ability to displace the radioligand. In either case, the basic binding assay is performed using the same basic method (Figure 2).
Kinase Phosphorylation Filter Binding Assays
Protein kinases regulate signal transduction by catalyzing the phosphorylation of target protein substrates, thereby altering the protein's activity. The protein kinase superfamily, containing 518 potential different enzymes,2 constitutes an important set of targets for drug discovery. In recent years, kinase inhibition has become a major area for therapeutic intervention, and high throughput screening has successfully identified a number of kinase inhibitors.3
High throughput screening using filter plates is a useful assay format to identify drugs that inhibit the phosphorylation of a target substrate. Filter plate and assay selection is based on the nature of the substrate molecule to be phosphorylated (e.g., a short synthesized peptide or a native protein). Depending on the type of substrate, two different methods of filter-binding assays are common: ion-exchange capture of peptides onto phosphocellulose filters or precipitation of protein substrates onto glass fiber filters.
Kinase assays using peptide substrates are best suited for assays on phosphocellulose filters. Phosphocellulose is highly negatively charged even at low pH. The kinase reaction is stopped by the addition of dilute phosphoric or acetic acid, which inactivates the kinase and simultaneously protonates any basic amino acids and the N-terminal amine of the peptide substrate.
The ability of phosphocellulose paper to retain the phosphorylated, positively charged peptides, while letting pass the negatively charged (g-33P) ATP, is the fundamental mechanism by which the two radioactive substrates are separated. Phosphocellulose filter binding assays are a proven, reliable, and automation-compatible methodology that directly measures phosphorylation.
The more typical filter-based assay that is used in conjunction with protein substrates and/or cellular extracts involves the use of glass fiber filter (GFF) to separate phosphorylated protein substrate from g-33P. The addition of trichloroacetic acid stops the phosphorylation reaction and precipitates the target protein substrate. The precipitate is captured on the GFF while the unincorporated (g-33P) ATP is not precipitated and passes through the filter.
Miniaturization of Filter Binding Assays
Filter-based receptor binding and kinase assays have been performed successfully on 96-well platforms. To increase throughput while reducing reagents, cost and time, the MultiScreenHTS 384-well filter plate has been developed and optimized for automated filter-binding assays. This 384-well format increases throughput of bioassays without sacrificing sensitivity, robustness, or precision.
The design is optimized for radiation detection and is compatible with coincidence scintillation counting directly in the filter plate. This makes it possible to achieve the same high sensitivity attained using a 96-well plate. Filter binding is directly scalable from 96- to a 384-well format, as shown using parallel experimental procedures, reagents, and receptor systems (Figure 3). The higher density of the 384-well filter plate allows for quantitative binding assays in a robust, fast, and reagent-saving format.
The smaller surface area of the 384-well filter plate significantly decreases nonspecific binding and allows for more efficient washing of the filter disc. The result of this is better well-to-well reproducibility when parallel assays are performed in 96- and 384-well formats.
Performing Assays Directly in the Filter Plate
A major goal in the optimization of any screening campaign is to reduce the number of required assay steps and manipulations. Kinase assays, as well as other enzymatic reactions, typically are performed in a coordinated manner such that the timing between reaction initiation and termination are carefully controlled. The fewer manipulations conducted, the greater the number of reactions performed in a given period of time.
The MultiScreenHTS384 enhances assay throughput by providing a high-density platform filter binding assay in which all the reaction steps are performed directly in the filter plate. This eliminates the extra step of transferring reactions from a separate reaction plate, and reduces the number of manipulations. Moreover, solid radioactive waste is also reduced.
In multiple side-by-side receptor binding and kinase assays, the performance of in plate and out of plate (i.e., when the reaction is carried out in a solid-bottom plastic assay plate and then stopped-reaction volumes are transferred to the filter plate) assays has been shown to be equivalent. Saturation, equilibrium, and kinetic assays can be carried out successfully using in-plate protocols
A pretreatment of the phosphocellulose media before performing kinase assays directly in the filter plate prevents potential interference from the filter.4 Figure 4 demonstrates the ability to perform quantitative kinetic experiments in this manner. By minimizing the surface area exposed to the reaction and pretreating the filter media, the differences between out-of-plate and in-plate assays are indistinguishable. The Km values determined in- and out-of-plate (13 and 17 mM, respectively) are typical of those reported previously.5
High throughput secondary screening to characterize specificity and potency of compounds that can affect signal transduction pathways is a key component of drug development in many therapeutic areas. MultiScreenHTS 384-well filter plates have been shown to be useful across a wide range of these applications. Both quantitative and screening type assays can be performed with the same robustness and reproducibility as has been frequently reported for the 96-well filter plate format. Bioassays in the 384-well filter plate can be performed with a reduction in both reagent amount and assay volumeresulting in a significant savings of both cost and time.
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