A. Gagne, E. Robitaille, M. Harvey, J.-A. St. Pierre, D. Wenham and P. Banks
High throughput screening is an indispensable tool for modern drug discovery1-3. The process is typically highly automated and can provide screening rates over 100,000 samples in a 24 hour period. To achieve this, assays must be simple, preferably mix and read type assays, where all reagents necessary to perform the assay are added sequentially to the microtitre plate without the need for transfer, aspiration, centrifugation or wash steps. In the same vein, the fewer the addition steps, the higher the screening rate achievable. Furthermore, assays must be miniaturizable in order to be adopted by most screening laboratories, since lower reagent consumption is an ever increasingly important attribute. Moreover, the use of radiometric detection tends to increase screening costs due to the need for significant safety and waste disposal costs, thus assays should be non-radiometric.
Arguably, fluorescence polarization (FP) detection is the best performer in that most of the ideal attributes for G-protein coupled receptor (GPCR)-ligand binding assay platforms (homogeneous, few addition steps required, miniaturizable, non-radiometric and ultra high throughput) are realized. There is no question that this assay is a simple non-radiometric mix and read type assay with few addition steps4-6. Ultra high throughput operation has been demonstrated for real screening applications7 and satisfactory performance with competitive economy has been demonstrated for the demanding application of GPCR ligand binding. Using red-shifted fluorescent labels, such as BODIPY-TMR, colored compound interference and autofluorescence from cell membrane fragments can be minimized7-9. Furthermore, nM binding affinities can be assayed with satisfactory accuracy and precision10.
The only insurmountable limitation to FP with the current level of HTS reader sensitivity, available fluorophores and labeling capabilities is that primarily, only peptidic GPCR ligands are suitable. Small molecule ligands below 1000 amu are difficult to label while preserving biological activity. Ligands greater than 5000 Da are not only difficult to label, but their size tends to preclude adequate assay performance. While size-independent detection is available with other non-radiometric detection technologies (FMAT, FIDA), they are still limited from a fluorescence labeling perspective. Furthermore, each of these detection technologies requires a dedicated reader only available from the detection technology provider. In addition, each of these technologies relies on scanning the contents of the well, which typically requires read times/well greater than one (1) second and thus precludes ultra high throughput operation at this time.
This application note serves to demonstrate the broad applicability of fluorescence polarization detection for peptidic ligand binding to GPCR targets. In addition to the eight ligands described here, fully optimized kits (pharmacology, assay performance, DMSO tolerance, kinetics and stability) are available. See [FP]2 brochure, reference number H78392, for more detailed lists.
Principles of Fluorescence Polarization Detection
The physical process that allows fluorescence polarization detection is centered on the principle that smaller molecules rotate faster than larger molecules in solution. Fluorescence can be used to probe these differences in rotation rates since the process of fluorescence, the time required for a fluorophore to emit a photon after excitation, also called the fluorescence lifetime, requires a measurable amount of time, typically in the nanosecond range for most fluorophores. If we assume that this time is typically a cons tant for a particular fluorophore, regardless of whether it is free in solution, derivatized to a peptide or, while covalently attached to that peptide, also bound to a receptor, one can differentiate between the three states by the extent of rotation that occurs over this time. The greater the rotation, the greater the depolarization of the excitation light leading to a smaller polarization signal.
In the case of a fluorescent derivative of a peptide bound to a GPCR embedded in a cell membrane fragment, little rotation can occur over the fluorescence lifetime since the cell membrane fragment behaves essentially as a solid phase over this period. Thus emission from the fluorophore remains polarized to a large degree (high signal). Conversely, if the derivatized peptide is displaced from the GPCR, as would be the case in the presence of a potent inhibitor to the ligand receptor interaction, significant rotation can occur over the fluorescence lifetime, which translates into a significant depolarization (small signal). In the intermediate cases of partially bound derivatized peptide, the magnitude of the signal is directly proportional to the extent of binding, to a first approximation10,11.
Materials and Methods
All fluorescently labeled peptides listed below in Table 1 are available from PerkinElmer Life Sciences. They are provided as off-the-shelf reagents accompanied by buffer recommendations, which have been optimized to allow for maximum assay performance. All have been tested against commercially available recombinant GPCRs expressed in mammalian cell membrane preparations.
Performance validation of the peptides consisted of verifying appropriate pharmacology by titrating the binding event with known displacers, usually the native peptide, and detecting the concentration-related response with fluorescence polarization detection. In addition to this, the suitability of each FP assay for transition into a primary screen was determined by evaluating the Z factor12 from multiple replicates of data. Z factors greater than 0.5 indicate robust assays, which can easily transition into a screen.
All assays were conducted in black, 384-well microtitre plates (Corning Costar, Cat. No. 3654), except for Bombesin (6-14) and Urotensin II, which used a different Costar plate type (Cat. No. 3710). The total assay volume was 40 μL. The order of sequential addition of reagents was as follows:
1. Add 20 μL of inhibitor or buffer
2. Add 10 μL of fluorescent peptide
3. Add 10 μL of membrane preparation
To achieve appropriate final concentrations of reagents, the stock solutions for plate loading of fluorescent peptide and membrane preparation were 4x concentrates and that for inhibitor was 2x concentrates.
Results and Discussion
Figures 2 and 3 demonstrate Apelin and Urotensin II pharmacology as examples for the peptides listed in Table 3. Both radiometric and FP data are demonstrated.
In each case, the pharmacology was compared to radiometric filtration assays and similar pharmacology was observed. This data is provided in Table 3 for these peptides and for Vasopressin and Bombesin(6-14) also.
An example of a scatter plot for Vasopressin binding to the V1b receptor subtype is provided in Figure 4. The high number of replicate data allows for an accurate assessment of the Z factor, which in this case is 0.51, which meets the criteria for "an excellent assay" in relation to screening as described by Zhang et al.12 The separation band12 between the two distributions, assuming Gaussian behavior, is greater than 40 mP.
Table 4 c ontains performance data obtained in a similar manner to Vasopressin for all the peptides listed in Table 1. In most cases, the assay window, defined as the difference between the Bound and Displaced signals shown in Figure 4 (Δ mP), is either approaching 100 mP or exceeding it. Furthermore, each of the assays demonstrates a Z factor > 0.5, indicating "an excellent assay" which can easily transition to a high throughput screen.
All fluorescently-labeled peptides listed in Tables 3 can be used to perform high throughput primary screening of compounds using fluorescence polarization detection. Appropriate pharmacology and satisfactory assay performance enables an easy transition to screening conditions. This simple and robust assay platform allows for ultra high throughput screening for GPCR binding applications.
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