Vronique Brechler, David Handfield, Martin Boissonneault, Esther Tremblay, Benoit Houle and Luc Mnard.
Recent advances in combinatorial chemistry and genomics programs have led to an unprecedented increase in both the number of compounds and potential drug targets that can be screened. The development of automated liquid handling and microplate assay systems has helped to meet the demand for increased throughput. Concomitantly, the introduction of homogeneous, non-separation, assay systems has eased the development of fully automated assays, especially for G protein-coupled receptors (GPCRs). GPCRs are composed of a large family of membrane-bound receptors that are characterized by the presence of seven hydrophobic membrane-spanning regions (Watson et al ., 1994). These receptors are involved in numerous aspects of cell signaling and regulation, and represent one of the major classes of the current therapeutic drug targets for the drugs sold today. Traditionally, pharmacological binding assays for these receptors have been performed using filtration assays that are difficult to automate.
For over ten years, PerkinElmer Life Sciences has been actively involved in the development of expression systems and novel assays for GPCRs. Given the current market need for simple homogeneous assays for GPCRs, PerkinElmer developed a novel line of reagents that are ready-to-use and pre-formatted for detection of ligand-receptor interactions. Using FlashPlate microplates as the homogeneous platform, we have developed a proprietary coating procedure to allow the stable and efficient immobilization of recombinant GPCRs on FlashPlate microplates. Assays are microformatted to a minimum volume of 25 μl, thereby reducing the costs of reagents and radioactive waste management, but can easily be formatted to 50 μl.
Membrane-coated FlashPlate microplates are suppli ed with the appropriate PerkinElmer radioligand, thereby making ScreenReady Targets a complete assay solution for high throughput screening (HTS) campaigns, focused library screening or small to large scale profiling (Figure 1). Twenty-one different ScreenReady Targets are currently available from PerkinElmer, one of which is the human motilin MTL-R1A receptor. Motilin is a 22-amino acid peptide secreted from endocrine cells of the small intestine that stimulates gastrointestinal contractility (Tonini et al ., 1996). The receptor for motilin has been cloned recently (Feighner et al ., 1999) and belongs to the GPCR family. Development of motilin receptor agonists and antagonists would be useful in the treatment of multiple disorders of the gastrointestinal motility. Here we present the pharmacological properties of the human motilin MTL-R1A receptor using the motilin ScreenReady Targets product.
Material & Methods
[125I]-motilin (2200 Ci/mmole) and 96-well FlashPlate Basic microplates were from PerkinElmer Life Sciences Inc.
Motilin was obtained from Bachem Inc. Membranes from CHO cells stably expressing the human motilin MTL-R1A receptor were used for the study.
Membranes from CHO cells expressing the human motilin MTL-R1A receptor were immobilized into 96-well FlashPlate Basic microplates using PerkinElmers proprietary coating procedure. Stability studies were performed using motilin ScreenReady Targets stored for various times at 4C in the dark. Homogeneous radioligand assays were performed in a volume of 25 μl.
Homogeneous Radioligand Binding Assay
Experiments were performed in binding buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.1% BSA) using a two-step procedure. Brie fly, 12.5 μl of binding buffer without (total binding determination) or with motilin at 1 μM final concentration (non-specific binding determination) were first added to membrane-coated FlashPlate microplates, followed by the addition of 12.5 μl [125I]-motilin. Incubation was performed at room temperature for 1 to 5 hours before counting on a PerkinElmer TopCount instrument (30 seconds/well).
Kd and Ki values were calculated using GraphPad Prism Software. Signal to background ratio (S/B) (total values/ nonspecific values) was calculated using a concentration of radioligand close to the Kd value.
Results and Discussion
Saturation Binding Assays
Membranes from CHO cells expressing the human motilin MTL-R1A receptor were immobilized into 96-well FlashPlate microplates and the affinity of [125I]-motilin for the receptor was determined. Saturation experiments using motilin ScreenReady Targets were performed on the day of membrane immobilization (Figure 2, A) or upon storage at 4C for three months (Figure 2, B). A similar single class of high affinity binding sites was detected from the saturation experiment performed on the day of membrane immobilization or after three months upon storage at 4C (Kd = 1.0 nM and 0.75 nM, respectively). Calculated Kd values were comparable to those previously reported (Feighner et al ., 1999; Thielemans et al ., 2001). Bmax values were also very similar in both assays (4.3 pmol/mg at t= 0 and 3.6 pmol/mg at t= 3 months). S/B values calculated at a concentration of radioligand close to the Kd value were comparable in both assays (5.1 vs. 4.6), showing that the immobilized motilin receptor maintained its pharmacological binding properties for at least three months at 4C.
Intra-plate variability of [125I]-motilin binding using motilin ScreenReady Targets was assessed. Membranes from CHO cells expressing the human motilin MTL-R1A receptor were immobilized into 96-well FlashPlate microplates and binding assays were performed using 48 wells for total binding determination and 48 wells for non-specific binding determination (Figure 4). The CV and S/B values led to a calculated Z factor (Zhang et al ., 1999) of 0.75, showing the robustness of the assay.
Using PerkinElmers proprietary coating procedure, we have developed ScreenReady Targets for homogeneous radioligand binding assays. The results obtained using the human motilin MTL-R1A receptor show that genuine pharmacological properties (Kd, Ki) can be determined and correspond to reported values (Feighner et al ., 1999; Thielemans et al ., 2001). Pharmacological analysis of the coated receptors showed that they maintain their binding properties, even after prolonged storage (3 months) at 4C. Low intra-plate variability (3.5%) on [125I]-motilin binding and a robust Z factor (0.75) demonstrate the usefulness of ScreenReady Targets for HTS assays.
ScreenReady Targets offer the following advantages:
Homogeneous assays with high reproducibility
Ready-to-use for radioligand binding assays
Stable for three months at 4C
Microformatted assays (25 μl or higher)
Resistant towards DMSO (1% - 5%)
Integration into automated systems such as PerkinElmers PlateTrak, MiniTrak and MultiPROBE II
Supplied with the appropriate PerkinElmer radioligand
ScreenReady Targets reagents have been developed for 21 GPCRs to date demonstrating the versatility and broad applicability of this new line of HTS reagents. Please contact us at firstname.lastname@example.org for an updated listing of all ScreenReady Targets available.
Feighner et al . (1999) Science 284, 2184-2188.
Thielemans et al . (2001) Brain Res. 895, 119-128
Tonini et al . (1996) Pharmacol. Res. 33, 217-226. Watson et al . (1994) The G-protein linked receptor Facts Book, ed. Academic Press, 1-427.
Zhang et al . (1999) J. Biomol. Screening 4, 67-73.
ScreenReady Targets poster presentation: Boissonneault M. et al . (2001) Development of the Motilin Receptor as a ScreenReady Targets for High Throughput Screening.