2000 MDC 4th International Drug Discovery Conf.
This year's International Drug Discovery Conference was a huge success, thanks to all of our presenters! The
presentation topics are listed below. For those presentations having a downloadable (pdf) version, we have
indicated the file size of the pdf.
characterization of receptors and cellular signalling
- FLIPR: A platform for orphan G-protein coupled receptor functional genomics
Dr. Bob Ames, SmithKline Beecham
G-protein coupled receptors represent the largest class of drug targets. While there are over 150 different subtypes of GPCRs with known ligands, there is an equally large number of orphan GPCRs for which the ligands are unkown. Orphan GPCRs identified through genomic and EST sequencing efforts represent a source of new drug targets. Using what has been referred to as a reverse pharamacology approach, we are using cells expressing the orphan GPCRs to search for ligands activating these receptors. A FLIPR based calcium mobilization assay is being used as a platform functional assay for this effort. This presentation will be an overview of the field of orphan receptor biology and will highlight several recent examples of orphan receptor ligand pairings accomplished on the FLIPR.
Download [PDF] (772 K)
- Highly specific CXCR4 antagonists with potent anti-HIV activity
Dr. Dominique Schols, Rega Institute
- Cross receptor interactions in metabotropic glutamate receptor functional assays: G-protein cross talk, and GPCR hetereodimers
Dr. Mike Johnson, Eli Lilly
Download [PDF] (356 K)
- Pharmacological characterization of 7TM receptors using FLIPR and chimeric/promiscuous G proteins— dependence of GABAB receptor ligand pharmacology on the co-transfected G Protein A-subunit
Dr Philip Szekeres, SmithKline Beecham
FLIPR has established itself as one of the major platform technologies for high-throughput screening of 7TM receptors. However, since FLIPR measures changes in intracellular calcium levels, it is best suited for screening 7TM receptors which are endogenously linked to Phospholipase C (PLC) and the mobilization of intracellular calcium. In order to study receptors linked to other signal transduction pathways, many groups have used promiscuous G protein a-subunits such as Ga16/Ga15 or chimeric G proteins such as Gqi5/Gqo5/Gqs5 in order to re-engineer the signal transduction pathways activated by these non PLC-coupled receptors and hence study their pharmacology on a FLIPR. One potential concern about using such articial systems is that the results obtained may not necessarily reflect those obtained using a more physiologically relevant system. This talk will focus on the strategies used in our group to screen orphan 7TM receptors for endogenous ligands on FLIPR using a combination of chimeric and promiscuous G proteins. In addition, potential pitfalls of using these systems for subsequent SAR analysis of receptor ligands will be discussed.
Download [PDF] (860 K)
- The combination of FLIPR and receptor alkylation (Furchgott analysis) to study receptor pharmacology
Dr. Rich Porter, Cerebrus
The use of FLIPR to study the agonist pharmacology of receptors is well established allowing rapid measurement of transient responses. Its use avoids many of the confounding issues of traditional second messenger assays such as receptor desensitisation following prolonged agonist exposure times.
The 5-HT2C receptor has been demonstrated to undergo RNA editing altering the efficiency of G protein – receptor interaction with a concomitant effect on agonist responses. Our studies have demonstrated that in addition to receptor density, the cell culture conditions, constituents of the dye loading buffer and cell density can also alter agonist responses at 5-HT2C receptors. In order to further examine the consequences of RNA editing on receptor function we have used a chemical alkylating agent to irreversibly antagonise a proportion of the receptors in order to examine the relationship between receptor occupancy and agonist induced responses.
Phenoxybenzamine (PBZ) is an irreversible antagonist of 5-HT2C receptors alkylating the active site and thereby preventing agonist induced receptor activation. Under conditions where the concentration of PBZ is sufficient to reduce the maximal 5-HT response by 40-70%, (eliminating receptor reserve) we determined the KA and relative intrinsic activity of agonists at the receptor.
Data will be presented on the methods and results obtained for both full and partial agonists at the 5-HT2C receptor (5-HT, mCPP, MK-212 and Ro 60-0175). In general, the data suggest that agonists are less potent and efficacious at edited 5-HT2C receptors than at the genomically encoded 5-HT2C receptor.
- The development of a mechanism-based Cytosensor (discontinued product) assay for anti-ischemic compounds
Dr. Craig Beeson, University of Washington, Seattle
The functional recovery of heart tissue following ischemia is correlated with the degree of mitochondrial damage and attendant metabolic impairment. The normal working heart derives much of its energy from the oxidation of lipids (65%) and, to a lesser extent, from glucose (30%). It has been found that if the heart metabolism is altered to lower the oxygen demand (i.e., less lipid oxidation), the degree of post-ischemia mitochondrial damage is reduced. Two compounds that reduce lipid oxidation in heart tissue, trimetazidine and ranolazine, have shown considerable promise as anti-ischemic drugs in clinical trials. While it is known that these compounds diminish b-oxidation of lipids in heart tissue, the mechanism of action and molecular targets are not known. We have developed a mechanism-based assay to study the effect of these compounds on rat heart myoblasts. The Cytosensor microphysiometer has been modified to directly measure the rate of lactate extrusion, oxygen consumption and glucose uptake. When these parameters are combined with the standard acid extrusion rates, the biochemical fluxes associated with energy metabolism can be determined with high kinetic resolution. It is proposed that this assay will enable the characterization of the biochemical mode of action for these compounds. This information should provide better insight into the potential molecular target for these novel anti-ischemic drugs.
Download [PDF] (228 K)
CLIPR™ chemiluminescence plate reader
- Evaluation of luciferase and SPA measurements in 384- and 1536-well format assays using the CLIPR
Dr. Dan Brown, Bristol Myers Squibb
The emergence of the 1536-well plate format as a routine high-throughput screening platform presents both challenges and opportunities to improve performance. We have evaluated the performance of the CLIPR with a luciferase reporter assay and conducted SPA proof of principle experiments in 384- and 1536-well format plates. We have also compared the results obtained with the CLIPR with those obtained from conventional plate readers that use a photomultiplier tube. From these evaluations, key factors affecting both assay miniaturization and detector performance with 384- and 1536-well formats have been identified.
- An evaluation of performance of various HTS luminescence readers
Dr. Tony Jurewicz, Smith Kline Beecham
The emergence of high-density plate formats for high throughput screening necessitates complex and expensive instrumentation. Thus, instrument evaluation has become even more critical for today's drug discovery efforts. Standard procedures are needed to evaluate instruments on a level playing field. These procedures must take into account each instrument's nuances and microplate preferences.
We have developed a standard protocol to evaluate detector performance and we are currently applying it to all our readers in order to facilitate the transition between assay development and HTS. In addition we are evaluating different platforms for ultraHTS (1536-well microplates and beyond) in order to make the right investments in the next generation of readers.
Luminescence is one of the detection modes better suited to work in miniaturized formats. As such we have tried to investigate how the new luminometers (imagers) work in comparison with traditional photo-multiplier tube based (PMT) readers. Data will be presented comparing the performance of CLIPR as a luminometer vs. other imagers and several PMT readers using 384-well plates.
Download [PDF] (696 K)
new applications and developments
- A novel resonance energy transfer assay for mitochondrial potential using FLIPR: Development of a high-throughput screen
Dr. Jim Dykens, Mitokor
Using FLIPR, we have developed a fluorescence resonance energy transfer (FRET) technique that permits real time monitoring of mitochondrial membrane potential in intact cells. The technique relies on energy transfer between the cardiolipin stain nonyl acridine orange (NAO), and tetramethylrhodamine methyl ester (TMR) that is sequestered by mitochondria as a Nernstian function of and concentration. FRET occurs when the dyes co-localize to the mitochondria resulting in quenching of NAO emission by TMR in proportion to DYm. Specificity of NAO for cardiolipin and requirement for close proximity of the dyes for FRET to occur eliminates plasma membrane potential as a confounding variable. Optimization of FLIPR operating parameters helps minimize dye concentrations; 80nM NAO and 150nM TMR yield sufficient signal with a monolayer of 60K SH-SY5Y neuroblastoma cells per well in a 96-well plate. Validation studies indicate that NAO is rapidly dequenched to its initial value by CCCP+ with EC50 = 2, and that hyperpolarization by oligomycin increases NAO quenching (EC50 = 0.5. The assay is adaptable to cells permeabilized by 0.01% digitonin and data are in accord with anticipated responses to well-described mitochondrially active agents such as bongkregic acid, cyclosporin A and ruthenium red.
- New reagent for FLIPR: an unmet need
Dr. Dieter Klaubert, Molecular Devices
The FLIPR instrument has found wide acceptance in the HTS/Drug Discovery arena mainly because it is a unique and enabling technology. However, this does not mean that improvements in both the instrument and its uses are not possible. MDC has embarked on a program to support its instruments with a reagent stream to further enhance their versatility. Some of the design considerations and results for these new reagents are presented.
Download [PDF] (2.1 MB)
- Practical use of JC-1 in a drug discovery setting
Dr. Per Arkhammar, Bioimage
JC-1 (5,5', 6,6’-tetrachloro-1, 1’, 3, 3'-tetraethylbenzimidazol-carbocyanine iodide) is a cationic dye
that preferentially accumulates in cellular compartments that have a negative membrane potential. This makes it
rather selective for active mitochondria. The mitochondrial membrane potential regulates the degree of accumulation
and it increases upon mitochondrial hyperpolarisation. Whereas the fluorescent probe in its monomeric form emits
green fluorescence, accumulation in mitochondria to concentrations in the millimolar range results in formation of
aggregates of the probe that emit red fluorescence. The ratio between green and red fluorescence can thus be used
as a dynamic measure of JC-1 accumulation in mitochondria and thereby the mitochondrial membrane potential. We have
used JC-1 to study mitochondrial function in a number of cell types. Recently we have successfully transferred the
measurements to FLIPR format using the double emission capability of the instrument. We have studied how several
known metabolic and respiratory inhibitors as well as ionophores affect mitochondrial membrane potential. The
mitochondria can, while displaying immediate sensitivity to ionophores transporting K+ or H+, under normal
circumstances compensate for a block of the glycolytic pathway, of the electron transport chain or of oxidative
phosphorylation so that it will not be detected as an immediate change in mitochondrial membrane potential.
A partial block of the electron transport chain and the ATPsynthetase makes changes in mitochondrial membrane
potential occur quicker or at lower concentrations of the tested substances. The JC-1 assay of mitochondrial
membrane potential can be used to detect desired and undesired effects of compounds on cell metabolism. Examples
include counter screens for unwanted effects of compounds selected for their primary effect on a target that is
either normally regulated, or partly regulated, through cellular metabolism or in itself a metabolic regulator.
Primary screening for compounds with a direct mitochondrial uncoupling activity in mitochondria may be another
use of the assay. Data from a test screen on pharmacologically active compounds will be presented.
Download [PDF] (644 K)
- Assay development of voltage- and ligand-gated ion channels on FLIPR using a novel membrane potential dye
Dr. Michael Xie, Millennium Pharmaceutical
Download [PDF] (572 K)
ion channel assays
- Development of a FLIPR based assay for HERG channel
Dr. Weimin Tang, Aventis
Many drug candidates have side effects leading to acquired long QT syndromes which are linked to the blockage of ion channels in ventricular myocytes. HERG channel underlies IKr current and has been reported to be blocked by many drugs including Dofetilide, Sertindole and Terfenadine. It will benefit for drug development if we can screen out compounds blocking HERG channel. Currently, the most common method for the detection is patch-clamping which is labor intensive and therefore limits the possibility to screen a large number of compounds in a short period of time.
In an effort to develop high-throughput screening method for HERG channel, we developed a CHO cell line stably expressing HERG channel, confirmed by patch-clamping. Using software STATISTICA design of experiment module, we have designed a FLIPR-based assay. The assay can detect Dofetilide at IC50=500nM and identify 5 out of 6 inhibitors at concentration of 1 mM. Our results demonstrated that a FLIPR-based HERG channel assay has been developed.
Download [PDF] (700 K)
- Vanilloid receptor pharmacology in recombinant and primary culture systems
Dr. Steve Brough, SmithKline Beecham
The vanilloid receptor 1 (VR1) is a ligand-gated ion channel which plays an important role in nociceptive processing. The rat vanilloid receptor 1 (rVR1) was recently cloned, stably expressed in HEK293 cells and pharmacologically characterised. VR1 is also endogenously expressed in rat dorsal root ganglion (DRG) cells. Production of rat DRG cultures is labour intensive and offers low yield thus is unsuitable to 96 well format. Using FLIPR384 affords the opportunity to compare the pharmacology of rat VR1 in our recombinant system with that of the endogenous receptor in primary cells.
Different relative efficacies of agonists were observed, suggesting varied receptor expression levels in the two cell systems, but the rank order of potency (RTX>capsaicin=olvanil>AM 404>anandamide) was identical. Antagonist affinities were comparable in the two systems.
In conclusion, we have developed a robust, reproducible assay in FLIPR384 which allows the characterisation of the rat VR1 receptor in a physiologically relevant primary cell system and shown that our recombinant system portrays similar pharmacology.
Download [PDF] (400 K)
- ASIC: proton-gated ion channels possibly involved in pain and neurodegeneration
Dr. Tino Jorgensen, Neurosearch
Download [PDF] (850 K)
- Characterization of vanilloid and purinoreceptors in an immortalized human sensory cell line using the FLIPR
Dr. Jan Smith, Advanced Medicine
- Ligand and voltage gated ion channels in TR14 neuroblastoma cells: functional characterization by FLIPR
Dr. Chris Grantham, Janssen Research Foundation
automation
- The evolution of FLIPR high throughput screening
Dr. Wil Lachnit, Roche Bioscience
Screening of pharmacological targets such as GPCRs and ion channels has evolved tremendously over the last decade. These screens typically consisted of radioligand binding filtration assays or radioactive ion flux assays in individual test tubes. Since that time, these assays have been miniaturized and automated increasing throughput from 100 to >5000 samples/day. However, binding assays as an example, are severely limited to targets that can be labeled with a radioligand. Furthermore, its utility is limited to generating hits that are antagonists. The introduction of FLIPR to the HTS arsenal has circumvented these limitations but, for it to be successful, a new infrastructure had to be created. Once in place, throughputs of 10,000 samples/day were achieved. With increased pressure to generate leads faster and more efficiently, additional HTS strategies have been identified and employed. These strategies have included implementation of FLIPR384 and incorporation of automation in areas such as biological reagent preparation and compound plate preparation. This evolution has significantly decreased costs and increased throughput to >40,000 samples/day, which has established FLIPR HTS as the preferred platform for GPCR and ion channel targets.
- Fully automated 384-well FLIPR assays— myth or reality?
Dr. Gary Allenby, Astra Zeneca
The demand for high throughput functional screens for G-protein coupled receptors (GPCRs) is ever increasing. Furthermore, combinatorial chemistry techniques are potentiating the number of chemicals tested per screen. The introduction of the 384 well FLIPR has addressed some but not all of these throughput demands. Resource constraints severely limit the effective use of FLIPR for screening, usually requiring two people to operate a FLIPR high throughput screen (HTS) at a rate of 150 to 200 microtitre plates/week. This throughput is inadequate given that a typical HTS contains approximately 3000 plates, translating to 20 weeks of screening, a large resource commitment. It is against this background that we have fully automated high throughput calcium assay screening on FLIPR384 using a robotic system designed to screen 24 hours/day seven days per week.
The system comprises a CRS 465 robotic arm on a 5 metre linear track. Instruments integrated onto the system include a tissue culture incubator with single plate random access 189 microtitre plate store, an Embla plate washer, Labsystems Multidrops, a Matrix Platemate, a high density compound storage unit, two further incubators and FLIPR384. The system was integrated by Scitec Automation in Switzerland using their scheduler (‘Clara’). After initial teething problems, the system has now performed a number of calcium GPCR-type assays, screening at a rate of approximately 100 plates per 24 hours, for 5 days per week. The automation of these assays has improved not only the throughput but also the quality of the data generated, with assay variability significantly reduced.
To maintain this throughput a number of further automation steps have been implemented, including the automation of cell seeding. It is likely that further automation of tissue culture will be required in order to maximise the potential of fully automated FLIPR384 screening. This will be discussed.
Download [PDF] (1.3 MB)
- Reality! A demonstration of automated high throughput FLIPR screening; data quality and quantity
Dr. Jenny Stables, Glaxo
The automation of FLIPR for high throughput screening has significantly increased not only the number of compounds tested in each G-protein coupled receptor (GPCR) screen but has also give us the opportunity to generate better quality and more consistent data. In order to achieve this improvement it has been necessary to implement a number of experimental approaches. These include the use of a reagent to negate background fluorescence and to reduce or eliminate washing steps associated with the use of Fluo-3, and the generation of more consistent data by using a ratiometric analysis of calcium response versus loading. Problems identified with FLIPR384 well dispensing which result in non-responsive wells will be discussed, detailing their cause and how to avoid them. In addition, metrics of an example screen will be discussed. These will include the validation of the screen using a sub-set of robustness compounds and a 10K pre-screen set of compounds in duplicate, together with a screening summary. The data will demonstrate the utility of automated screening in comparison to manual screening.
Download presentation [PDF] (844 K)
- Automated 384-well FLIPR-assays in HTS
Dr. Annika Jeppsson, Astra Zeneca
There has been an enormous increase in the demand for functional FLIPR assays in High Throughput Screening (HTS) during the last years. However throughput on the FLIPR has always been very limited. After the development of the 384 FLIPR, the next challenge on the way to improve throughput was to integrate the reader into an automated system. The benefits in workforce savings and throughput are obvious provided that the quality of the data is remained in an automated system.
We chose a Beckman/Saigan-system to automate the FLIPR assays using an Orca robotic arm on a system designed around a linear 2-metre track. Various instruments are integrated along the track to perform the assays
Our experiences of the integration have been positive. After start up problems with both hardware and software we are now running an average of 100 plates/day with maintained data quality.
|
