Thanks to everyone who attended our 2007 FLIPR Drug Discovery Forum, and a special thanks to our distinguished speakers.
If you missed this event, you will find several of the presentations available below for downloading. If a presentation is not available here, please contact the author directly.
- Validation of Different FLIPR Calcium No-Wash Dyes for Performance and Compound Interference Through Millipore's GPCR Profiler™ Service
Matthew Hsu, Ph.D., Millipore
GPCRs are important drug targets for HTS as well as selectivity profiling. Millipore's GPCR Profiler™ service is the first complete functional cell-based assay platform that uses a common validated calcium readout for over 130 GPCR targets across all three classes and 50 ligand families. It leverages the advantages of Millipore's proprietary ChemiScreen™ expression technology, which funnels all GPCR signaling through robust calcium mobilization for real time FLIPR assay without having to co-transfect any G proteins.
Using FLIPR calcium no wash dyes simplified the assay setup, and increased the throughput and reproducibility of the service. Not all commercial calcium no wash dyes, however, deliver the same result in terms of signal robustness, background, receptor sensitivity, quencher interference with color or auto-fluorescent compounds, and sometimes even the EC50 of the ligand for the same receptor, especially when suspension cell lines were used. The performances of 4 different calcium no wash dyes, including MDC's FLIPR calcium 4 assay kit, were compared to regular Fluo-4 calcium dye using different compounds and GPCR targets throughout Millipore's GPCR Profiler™ service.
- High-Throughput Screening for Allosteric Modulators of a GPCR using the Calcium-4 dye on FLIPR
Theresa Truitt, Hoffmann La Roche
Traditional HTS approaches to the screening of GPCRs look at a compound's ability to interact at the orthosteric ligand binding site. Recently, interest has turned to looking at alternative (allosteric) sites as potential drug candidates. A known GPCR that is implicated in feeding behavior was cloned into CHOK1 cells for use in a Calcium flux-based high throughput screening campaign. After evaluation of various Ca dyes from different manufacturers, the Calcium-4 dye from Molecular Devices was selected for the HTS effort. The Fluorometric Imaging Plate Reader 384 was used to detect ligand induced intracellular calcium release for evaluation of our compound library. Dye loaded cells were exposed to compounds followed by challenge with an EC10 of the known ligand. Compound-induced changes in either the potency (leftward shift of an agonist dose response curve) or efficacy (magnitude of the calcium release) were monitored and used to characterize hits. The optimized assay was successfully used to evaluate an 800,000 + compound library. Assay specifics such as robust Z' prime, hit rate and agonist response will be discussed.
- Optimization of Calcium Flux Mediated by the _4_2 Neuronal Nicotinic Acetylcholine Receptors Utilizing the FLIPR Calcium 4 Assay Kit
Jenny Zhang, Ph.D., Targacept, Inc.
The rapid procedure of the FLIPR Calcium 4 Assay makes it desirable in high throughput screening for potential ligands gating calcium (Ca) ion channels. Here, we report a successful application of this assay to detect Ca flux mediated by neuronal nicotinic acetylcholine receptors (nAChR) in SH-EP1 human epithelial cells expressing α4β2 receptors. Using HBSS/HEPES buffer, little Ca flux was observed in response to nicotine (Nic) up to 100 µM. Addition of 2.5 mM probenecid resulted in no enhancement on Ca response. By switching to a low sodium and high Ca buffer, MEBSS, the Ca response to Nic was amplified markedly. At 10 µM, Nic induced a 3.12±0.33-fold increase of peak Ca flux above baseline in MEBSS buffer, comparing to only a 1.31±0.12- fold response in HBSS/HEPES. Cell seeding density and cell culture time also affected the Nic-mediated Ca response. The Nic-mediated Ca flux was not affected by 1 µM atropine treatment. These nicotinic responses were competitively inhibited by an α4β2 nAChR antagonist, dihydro-β-eryoidine (DHBE); and concentration-dependently inhibited by an open channel blocker, mecamylamine (MECA). In addition, the EC50 and the maximal responses (Emax) collected from the FLIPR Calcium 4 Assay for about 70 potential nAChR ligands were compared to those from a Rubidium flux assay. There were significant correlations for both EC50 and Emax between these two assays. Thus, the FLIPR Calcium 4 Assay may serve as a rapid and reliable measure of functional activity of α4β2 nAChRs in SH-EP1 cells.
- Multispan GPCR Drug Discovery Platform — Taking GPCR Expression to the Next Levels
Tiina Sepp, Ph.D., Multispan, Inc.
G Protein-Coupled Receptors (GPCRs) are the most validated class of "druggable" targets due to their access to important pathways and their success in the marketplace. Because of the structural and functional commonalities among this large family of receptor proteins, it is critical and challenging to deconvolute the specificity of small molecule and antibody drug leads and antibody reagents for research and diagnostics. To address those needs, leveraging its robust GPCR expression technology and large collection of 320 full-length GPCRs, Multispan has established 3 versatile platforms to profile specificity of small molecule and antibody leads: 1) 90 stable GPCR functional assays, 2) 60 GPCR antibody profiling panels and 3) 70 transiently transfected GPCRs as a functional Ca++ assay panel.
Functional Ca++ flux assays are widely used for GPCR activation studies. Several commercial kits facilitate no-wash protocols that are considered to be more robust than those requiring wash steps. We evaluated the performance of four different commercial kits to obtain maximal signal to noise ratio as well as maximal sensitivity for a large functional screening panel of different GPCRs. Using the FlexStation, we compared responses in 48 GPCR functional assays using a subset of Multispan's GPCR compound profiling panel. Our data demonstrated superior performance of the new masking dye-based Calcium kit, Ca4, giving a DeltaF/F0 >0.75 for 5 times as many GPCRs as did the Fluo-4 NW kit.
The results of these experiments as well as data from additional GPCR small molecule and antibody profiling experiments will be presented. In one set of experiments forty compounds were tested as agonists to the FFA receptor. In the second project, a ligand was tested against a panel of 40 GPCRs and an unexpected off-target response was discovered. Multiple other samples of profiling experiments of different compounds will also be presented.
- Introduction of the FLIPRTETRA Aequorin Option for Luminescent Calcium Mobilization Assays
Jennifer McKie, MDS Analytical Technologies (Molecular Devices)
Use of photoproteins such as aequorin to monitor calcium mobilization has increased in popularity in drug discovery. Assay performance and reduction in costs are the main drivers for converting to this technology. FLIPRTETRA builds on its robust ability to perform calcium flux assays by offering a novel camera technology and cell suspension reservoir specifically designed to address the aequorin application. Using a single camera, aequorin luminescence can be monitored in addition to traditional fluorescence calcium mobilization assays. The new cell suspension reservoir, in conjunction with proven FLIPR® pipettors, can be used to perform suspension experiments in up to 1536-well format. Data is presented demonstrating performance of aequorin assays in adherent and suspension modes with the new aequorin option for the FLIPRTETRA.
Please contact Jennifer McKie for more information: jennifer.mckie@moldev.com
- Novel CNS Signaling Paradigm in Animal Models: Potential Implication for Drug Discovery
Marc G. Caron, Ph.D., Duke University Medical Center
G protein-coupled receptors (GPCR-7TM) mediate numerous physiological responses. These responses are regulated by kinases (GRKs) followed by binding of the phosphorylated receptors to the scaffold proteins, β-arrestins. This interaction not only dampens G protein-dependent signaling but also targets receptors for internalization and initiates additional signaling events through the ability of β-arrestins to scaffold signaling complexes. Our ability to visualize in real-time and in single cell these interactions has greatly facilitated our understanding of the molecular determinants and biological relevance of these interactions. In the brain the dopaminergic system is presumably implicated in the control of locomotion, cognition, emotion and affect as well as reward mechanisms. In mice, activation of D2-like receptors by pharmacological agents or genetic manipulations modulates not only the cAMP-dependent signaling pathway but also engages an Akt-PP2A-GSK3 signaling pathway. This latter effect is mediated by the ability of the receptor/β-arrestin2 complex to scaffold these proteins. Mice lacking a functional β-arrestin2 gene show diminished dopaminergic responses providing confirmation for the paradigm that G protein-independent signal transduction of monoamine receptors mediates behavioral outcomes. Accordingly, GSK3β+/- mice show diminished responses to the psychostimulant amphetamine and lithium, a direct inhibitor of GSK3, antagonizes dopamine-dependent locomotor responses in animals. Interestingly, in addition to its direct action on GSK3, lithium disrupts the complex of β-arrestin2-Akt-PP2A without affecting other functions of β-arrestin2. These findings suggest that selective disruption of D2R-β-arrestin2-Akt-PP2A complexes might represent an interesting drug target.
Another example where this type of signaling may play an important role is with the GPCR-7TM-like protein, Smoothened, the activity of which is controlled by Hedgehog proteins through their interaction with Patched. This signaling mechanism mediates developmental processes, cellular proliferation and differentiation through activation of the transcription factor Gli. Loss-of-function mutations in the Hedgehog/Pathched/Smoothened signaling pathway produce developmental phenotypes whereas gain-of-function mutations are found in many forms of cancer such as basal cell carcinoma and medulloblastoma. In zebrafish knock-down of either β-arrestin2 or GRK2 expression using stable "morpholinos" results in embryonic lethal phenotypes where the treated fish develop 'U-shaped' somites and many other defects that recapitulate loss-of-function of Hedgehog signaling. In cells GRK2 phosphorylates Smoothened and β-arrestin2 interacts with Smoothened and both GRK2 and β-arrestin2 contribute positively to pathway activation.
GPCRs are targets for a majority of therapeutic drugs currently on the market. However, these drugs have all essentially been discovered using the classical paradigm of GPCR signaling. The ability to monitor in real time and in live cells agonist-dependent 7TM-GPCR/β-arrestin2 interactions represents an attractive means of identifying novel compounds that can target receptors for which no therapeutic agents exist or functionally selective compounds that can block unconventional signaling pathways.
- The CellKey™ System - A Biorelevant Cell Based Assay Platform for Drug Discovery Applications
John Proctor, Ph.D., MDS Analytical Technologies (MDS Sciex)
Cell surface receptors and their associated signaling pathways are attractive drug targets. Drug discovery programs for many receptor types including GPCRs and TKRs rely on a variety of assays that traditionally involve radioactive, fluorescence and bioluminescence-based detection. The CellKey™ System is a revolutionary label-free assay platform that enables detection of endogenous receptor activation in its most biorelevant setting. We present data that illustrates the universal ability of the system to measure, in real-time, multiple endogenous cell surface receptor families in a single assay with minimal assay development. We compare the CellKey™ System to other cell-based assays and show its use in analyzing endogenous receptor pharmacology in adherent and suspension cell lines and primary cells. Through dissection of signal transduction pathways, we correlate the CellKey™ response to cellular physiology and identify the G-protein coupling mechanism of uncharacterized GPCRs. Lastly, we show examples of how the CellKey™ System's information-rich data and its ease of use can significantly reduce assay development and costs in drug discovery.
Please contact John Proctor for more information: john.proctor@sciex.com
- A Discovery Program for Better Taste: Screening for TRPM5 Ion Channel Modulators using Membrane Potential and Calcium Dyes with the FLIPRTETRA
Robert W. "Rusty" Bryant, Ph.D., Redpoint Bio Corporation
Discovery of new compounds to modulate human taste is driven by several commercial needs including improved low calorie sweeteners and compounds to block the bitter, unpalatable tastes of pharmaceuticals. TRPM5 is a calcium-activated monovalent cation channel which is a critical component of the bitter, sweet, and umami (savory) taste GPCR signaling pathways. In order to identify potent, selective modulators of TRPM5, we developed a high throughput FLIPR-based assay using recombinant human TRPM5-expressing HEK cells, membrane potential (MP) and Ca++ fluorescent dyes. ATP-activation of endogenous P2Y receptors elicited strong Ca++ response in both recombinant hTRPM5/HEK and parental HEK293 cells. However, ATP elicited MP responses only in hTRPM5/HEK cells, confirming the Ca++ dependence of TRPM5. A random library of 84,000 compounds was screened at 10 uM for TRPM5 modulators. Both enhancers and inhibitors were found at a relatively high 0.7% hit rate each. A robust specificity process was implemented to eliminate blockers of Ca++ and KCl depolarization responses. A calcium-triggered patch clamp assay confirmed the specificity and potency both TRPM5 enhancers and blockers. A number of selective TRPM5 enhancers and inhibitors were identified and are now in lead optimization.
- Comparison of Three Different Dye Systems at a Voltage-gated Potassium Channel and a Ligand-gated Ion Channel Using FLIPR384 and FLIPRTETRA
Karen Cook, M.S., Pfizer Global Research & Development
Ion channels are challenging targets for drug discovery efforts. Recent developments in both FLIPR technology and fluorescent dye systems have enabled more comprehensive and target-specific channel studies. The LED light source in the FLIPRTetra brings diversity and flexibility to experimental design. The FLIPRTetra can specifically excite a range of fluorophores and can detect fluorescence changes at multiple emission wavelengths simultaneously, allowing the use of FRET dye pairs, such as the Voltage Sensor Probe™ technology from Invitrogen. Both Voltage Sensor Probes™ and the Membrane Potential Dyes™ from Molecular Devices produce direct measurements of ion channel activity, as opposed to more traditional calcium-sensitive dyes, so that data can be more closely compared to in vitro electrophysiological measurements. The new Thalkal Dye™ from Molecular Devices differs from the Voltage Sensor Probes™ and Membrane Potential Dyes™, as it specifically measures the activity of potassium channels. This technology consists of a thallium-sensitive dye, and it utilizes the permeability of potassium channels to thallium ion. If the extracellular thallium ion concentration is appropriate for the channel of interest, opening of potassium channels will result in influx of thallium ion, leading to fluorescence of ThalKal™. This method directly measures changes in potassium channel permeability and may provide more clarity about potassium channel activity than voltage- and membrane-sensitive dyes. The present work compares the use of the three dye systems described above in development of an assay for a voltage-gated potassium channel, using both the FLIPRTetra and the FLIPR384. The Voltage Sensor Probe™ technology was also compared to the Membrane Potential Dyes™ for use with a ligand-gated ion channel.
- High Throughput Screening of Anion Channels with the YFP variant F46L/H148Q/I152L using FLIPR
Mark W. Thompson, B.S., Bristol-Myers Squibb
Chloride (Cl-) fluxing anion channels are demonstrated molecular targets for drugs used in the treatment of neurological affective disorders and cystic fibrosis, as well as being perceived as important therapeutic targets in other disease conditions. Although traditional screening approaches, such as radioactive anion flux, can readily be used to screen and profile the activity of compound libraries at anion channels, Bristol-Myers Squibb (BMS) has reduced to practice a robust functional high-throughput measurement of anion channel activity using a 384-well FLIPR. The approach is based upon a variant of the mutated yellow fluorescent protein (YFP-F46L/H148Q/I152L) stably co-expressed with target(s) of interest in HEK 293 cells. The technique offers high sensitivity, reproducibility and amenability to traditional high throughput screening platforms and has the added advantage of not requiring fluorescent dyes. We have applied this technique to Cl- channels to identify novel modulators from within the entire BMS screening deck. In addition, we have also applied the technique to early profiling of potential Cl- channel mediated toxicity. As part of our validation of the halide sensor technique, we have demonstrated a strong concordance between potency and efficacy determinations using automated patch-clamp and the halide sensor assay. These data presented illustrate the utility of the halide sensor assay in hit identification, lead optimization and lead profiling.
- A Novel Fluorescent Assay for Neurotransmitter Transporters
Phillip Jones, Ph.D., Sepracor
We have evaluated a novel neurotransmitter assay reagent from Molecular Devices that utilizes a fluorescent substrate for the biogenic amine transporters. Current assays use radioactive substrates and are therefore not the preferred method for large scale screening campaigns. In contrast, this homogeneous, no-wash assay is amenable to high-throughput screening. The KM for the substrate, determined using HEK293 cells stably expressing the recombinant human transporters for dopamine , norepinephrine, or serotonin, is 1˜2 micromolar for all three transporters, and at one micromolar substrate, the rates of transport provide signal:background greater than 9-fold within a 30 minute assay time. Hence, this substrate (unlike previous fluorescent probes) provides a robust and sensitive method to identify and characterize inhibitors at each of these transporters. The signal can be determined at assay end-point or in kinetic mode, and is linear up to at least 30 minutes. Z'-factors of 0.64-0.72 demonstrate the assay's reproducibility. IC50 values for a series of nine inhibitors were in reasonable agreement with those obtained using radiometric assays (R2 > 0.88 for each correlation). The IC50 values were constant at times from 15-45 minutes. In summary, the simplicity, adaptability and reproducibility of this fluorescent assay will facilitate the study of biogenic amine transporters in both academic and industrial settings.
