2003 MDC 7th 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.

novel applications using FLIPR® and FlexStation®

• Using FLIPR with Fluo-3 and Calcein AM to characterize AMPA/Kainate receptors in cerebrocortical neurons
  Thomas F. Murray, Ph.D., University of Georgia College of Veterinary Medicine

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  AMPA receptors lacking the GluR2 subunit are highly permeable to Ca²⁺ and may contribute to excitotoxicity. We have previously shown that activation of AMPA receptors is necessary for triggering spontaneous calcium oscillations in neocortical neurons. Approximately 8-15% of neurons in neocortical cultures express calcium permeable AMPA receptors. Agonist stimulated cobalt uptake is routinely used to identify these neurons immunocytochemically. Using FLIPR, we have measured cobalt uptake in murine neocortical neurons in an effort to quantify ion influx through Ca²⁺ permeable AMPA/kainate receptors. We used the dye calcein-AM whose fluorescence is rapidly and stochiometrically quenched by divalent metals including Co²⁺, while being only minimally affected by variations in intracellular Ca²⁺. Similar to past studies that have used different techniques, a basal level of cobalt influx was detected in neocortical neurons. Application of AMPA in the presence of cyclothiazide led to a concentration-dependent increase in cobalt uptake in the neocortical neurons. Similar concentration-dependent increments in cobalt uptake were seen with kainic acid treatment. NBQX, an AMPA/kainate receptor antagonist, blocked the AMPA-induced cobalt uptake which further indicates that cobalt flux occurs primarily through AMPA/kainate receptors. In conclusion, this fluorometric method affords a rapid and quantitative procedure for investigation of drug interaction with calcium permeable AMPA/kainate receptors.

  In cerebellar granule neurons loaded with the Ca²⁺ sensitive dye Fluo-3, domoic acid produces a rapid and concentration-dependent increase in [Ca²⁺]_I. Unlike the response in cerebral cortical neurons expressing Ca²⁺ permeable AMPA/Kainate receptors, the increase in cytoplasmic [Ca²⁺] in cerebellar granule cells is derived from the indirect activation of NMDA receptors, L-type Ca²⁺ channels and the reverse mode of operation of the Na+/Ca²⁺ exchanger. These studies with intact neurons demonstrate the utility of FLIPR in delineating mechanisms of glutamate receptor regulation of Ca²⁺ signaling.

• Allosteric modulation of metabotropic glutamate receptors
  John Dunlop, Ph.D , Wyeth Research

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  Metabotropic glutamate receptors represent an important family of glutamate-activated G-protein coupled receptors coupled to changes in intracellular second messenger levels and ultimately regulating neuronal function and excitability. Eight subtypes (mGlu receptor 1-8) have been identified and these have been sub-classified based on sequence similarity and principal signal transduction pathway. Initial drug discovery efforts targeting mGlu receptors were primarily focused on agents acting at the glutamate recognition site leading to the identification of subtype selective agonists and antagonists. More recently, several groups have described the identification of positive allosteric modulators of mGlu receptors 1, 2, 4 and 5. These agents act at sites distinct from the cytoplasmic glutamate-binding site near the transmembrane domains. Cell based functional assays on FLIPR are well suited to the study of allosteric modulation, in fact in a single assay agonism, antagonism or modulation can be evaluated. Recently, we developed a cell line co-expressing mGlu receptor 4 and a calcium pathway facilitating G-protein chimera to permit functional studies on FLIPR. In this presentation, recent data on allosteric modulation of mGlu receptor 4 will be presented.

• Development and use of human neural stem cell lines for functional pharmacology studies
  Kenneth Pollock, Ph.D., ReNeuron

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  RenCells are immortalised human neural stem cell lines that have been developed and characterised for their utility in a range of neuroscience research and development applications. These lines are derived from first trimester cadaveric foetal brain tissue obtained under national ethically approved protocols. Following retroviral transduction of primary cells with with an immortalising transgene namely v-myc or c-myc, lines were selected out using antibiotic resistance markers. Previous lines have also been derived using the temperature sensitive T antigen (tsT), but such lines are prone to genetic instability and rapid senescence in contrast to myc lines. Both tsT lines and RenCells are grown as monolayers on laminin coated flasks and are expanded in the presence of growth factors EGF and bFGF. Under these conditions the cells grow as an even monolayer with a polygonal morphology. Phenotypically they are ubiquitously nestin (neural stem cell marker) positive with a low incidence of beta-III tubulin (neuronal marker) and GFAP (astrocyte) positive cells. However under defined conditions, e.g. withdrawal of growth factors and/or addition of retinoic acid, RenCells undergo phenotypic (beta-III tubulin positive) and morphological changes, characteristic of more mature neurones. The growth characteristics of our neural stem cell lines make them ideal for multiwell plate formatted assays such as those developed for FLIPR and FlexStation.

  The cortex derived tsT line "Oggy" was profiled on FLIPR using Fluo-4. This cell line responds to Histamine and ATP but not to Carbachol. In these cells the histamine response is selectively blocked by Mepyramine (IC50 5nM). More recently RenCell VM (ventral mesencephalon) and RenCell CX (cortex) lines were profiled on FlexStation for both calcium changes (Calcium-3) and membrane potential changes. Undifferentiated RenCell VM cells responded to a range of agonists with a robust change in calcium; Carbachol = ATP>Histamine>N-Methyl dopa> NPY > 5-HT=NA. Similar activity was seen in differentiated RenCell VM cells. For membrane potential studies only KCL caused significant depolarisation. Both undifferentiated and differentiated RenCell CX responded with robust changes in calcium to Thrombin >> Carbachol=ATP>> Histamine. Only KCL caused significant depolarisation of these cells. Clearly GPCR coupled calcium signalling mechanisms are functioning in these lines. What remains to be explored more fully is the incidence of ion channel coupled receptors. We have recently carried out patch clamping analyses on differentiated RenCell CX cells which indicate these cells to have a heterogeneous spread of membrane potential and synaptic currents, but to date no action potentials. Further work is in progress to enhance the electrophysiological properties of these cells in culture.

• Comparison of FLIPR384 and FlexStation 96 and 384, using membrane potential assay and calcium flux assay
  Lone Pridal, Ph.D, Novo Nordisk A/S

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  The FlexStation 384 was tested to determine if it could be used for assay development and validation of HTS assays before transfer to the FLIPR. The tests were performed using two different assays, a membrane potential assay and a calcium flux assay.

  The pipetting CV was found to be: FLIPR-384 format, 0.3 µl DMSO dispense: 6-10%. FlexStation 96 format, 1 µl DMSO dispense: 4-6%; 384 format, 1 µl DMSO dispense: 1-3% and 384 format, 0.3 µl DMSO dispense: 3-5%.

  Calcium flux assay	Format	Sample volume µl	Intra- assay CV	Inter- assay CV	Z'	EC50 fold variation
  FlexStation	96	1	5	10	0.6-0.8	3-4
  FlexStation	384	1	5	10	0.4-0.6	2-3
  FlexStation	384	0.3	5-10	10-15	0.7-0.9	2-3
  FLIPR	384	0.3	5-10	10-15	0.6-0.8	2-3

  Membrane potential assay	Format	Sample volume µl	Intra- assay CV	Inter- assay CV	Z'	EC50 fold variation
  FlexStation	96	1	5-10	10-20	0.5-0.8	3-4
  FlexStation	384	1	10-20	15-20	0.2-0.6	4
  FlexStation	384	0.3	5	10	0.5-0.8	3-4
  FLIPR	384	0.3	10-20	15-20	0.5-0.7	3-4

  The FLEXstation 384 was found to be a reliable instrument for low and medium throughput applications.

• Opiod receptor regulation of intracellular calcium levels: a direct activation of phospholipase Cβ₃?
  Ping Yee Law, Ph.D., University of Minnesota

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  Although opioid agonist induced mobilization of intracellular Ca²⁺ has been observed in several cell models, the use of intracellular Ca²⁺ fluorescence to monitor the receptor activation has met with difficulties. In the past, the chimeric Gq/Gi α-subunit of the heterotrimeric G protein is utilized to assay for the activation of GPCRs such as opioid receptors that are coupled to the pertussis toxin-sensitive Gi/Go proteins. In current studies, based on the observation that Gq coupled-receptors could potentiate the effects of Gi/Go coupled receptors, we report a direct measurement of intracellular Ca²⁺ increase as a result of the opioid receptor activation of the Gi/Go proteins. Using the FlexStation to monitor the intracellular Ca²⁺ increase, we observed that activation of the µ-opioid receptor expressed in HEK293 or Neuro2A cells did not result in the Ca²⁺ fluorescence increase. Meanwhile activation of the P2y receptor with agonists resulted in a dose-dependent increase in the intracellular Ca²⁺ fluorescence. Interestingly, when opioid agonist was added to the HEK293 cells after the ATP response returned to control level, an opioid agonist concentration-dependent increase in the intracellular Ca²⁺ fluorescence was observed. The opioid agonist response required the activation of the P2y receptor, and appeared to be threshold-dependent. Since the bg-subunits of the heterotrimeric G proteins have been implicated in the opioid agonist induced increase in the intracellular Ca²⁺, it is tempting to hypothesize that bg-subunits from the Gi/Go bind to the PLCß3 thus potentiating the Gq α-subunit activation of this enzyme. However, the over-expression of either the control or the constitutive active Gq α-subunit did not result in a robust opioid agonist response. Subsequently, we determined that the PKC inhibitor GF109203X could attenuate the opioid response. The target of PKC remains unknown. Nevertheless, the activation of Gq-coupled receptor such as P2y could result in the ability of Gi/Go coupled receptor to mobilize intracellular Ca²⁺, and such paradigm could be used for high throughput assays of the Gi/Go coupled receptor activation.

• Design and validation of a functional assay of pancreatic K-ATP channels using FlexStation and patch techniques
  Simon Xinmin Xie, Ph.D., Deltagen Inc.

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  ATP-sensitive K⁺ (K-ATP) channels in pancreatic β-cells regulate the membrane potential and consequently modulate the insulin release. In an attempt to establish a high-throughput (HT) functional assay of K-ATP channel activity, we employed a fluorescence-based membrane potential assay in native pancreatic β-cell lines using a FlexStation microplate reader. Furthermore, we used the standard patch clamp technique to record these cultured b cells and acutely dissociated mouse pancreatic cells to validate this assay. We used four standard K-ATP channel modulators, and found there is a good correlation of drug potency measured by the HTS assay and the patch clamp technique. We first used RT-PCR to confirm that Kir6.2/SUR-1 subunits comprising the K-ATP channels principally express in these pancreatic β-cells. We then used the FlexStation to detect the membrane potential of the β-cells in response to K-ATP channel modulators. Two standard K-ATP channel blockers, tolbutamide and glibenclamide, and two openers, diazoxide and the novel diazoxide analog, 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216) caused concentration-dependent depolarization and hyperpolarization, respectively. Comparison of the four K-ATP modulators’ potency (EC₅₀ or IC₅₀) determined by FlexStation or membrane potential and input resistance changes using patch clamp recordings indicates a good correlation between the HT method and standard technique. Therefore, the 96-well format of fluorescence-based membrane potential assay is suitable for functional screening of compounds that potentially interact with K-ATP channels.

• Identifying agonists, antagonists and modulators of GPCRs using a single assay protocol with FLIPR
  Robert Bostwick, Ph.D., AstraZeneca

  Assay protocols that enable the detection of multiple types of pharmacological agents (agonists, antagonists and modulators) in a single experiment can leverage the efficiency of an HTS campaign by reducing screening time and consumption of reagents and compounds. Cell-based assays are amenable to this approach because they allow for measurement of different effects of test compounds on a functional response. Measurement of changes in intracellular calcium using the FLIPR is particularly well suited for this purpose because it is possible to measure a cellular response in real time following multiple compound additions. Example protocols for FLIPR cell-based assays used to detect agonists, antagonists, and modulators of a GPCR in either a sequential or simultaneous manner will be described. Assay validation and HTS performance for an HTS case study will also be presented.

• Using Molecular Devices Calcium 3 kit to do small molecule drug discovery at Athersys, Inc: it’s all the RAGE
  Robert W. Mays, Ph.D., Athersys, Inc.

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  Athersys, Inc. is a biopharmaceutical company founded in 1995, whose mission is to be a leader in the identification and development of therapeutics that extend and enhance human life. Athersys is currently utilizing its novel platform technologies, either separately or in an integrated strategy, to develop small molecule drugs to portfolios of key targets within defined therapeutic areas. The company’s platforms are: RAGE (Random Activation of Gene Expression), MAPC (Multi-potent Adult Progenitor Cells), GECKO (Genome-wide Cell-based Knock-Out) and SMCs (Synthetic Micro-Chromosomes).

  To date, the RAGE technology platform has been utilized to generate numerous cell lines expressing Validated Targets (VT) of therapeutic relevance for drug screening. By using the Molecular Devices FLIPR-384 and Molecular Devices Calcium Plus Kits, we have successfully completed HTS campaigns against 4 different RAGE-VT cell lines. Critical components of assay development prior to successful HTS are defining the optimum conditions for cell culture, plating and dye loading, thereby ensuring suitable Z’ values. We compared Molecular Devices’ newest calcium indicator dye product, the Calcium-3 Kit, to the Molecular Devices Calcium Plus dye, “No Wash” Dye, Fluo-3 and Fluo-4 calcium indicator dyes. We tested the dyes side-by-side against 6 different RAGE-VT cell lines expressing either chemokine, neurotransmitter or peptide activated GPCRs, or a ligand-gated ion channel. Our results indicate that the Calcium-3 dye consistently performed as well as, and in most cases, better than the other dyes in regards to maximal fluorescent signal and Z’ values. As a result of this comparison, all small molecule discovery programs at Athersys, Inc. are using Molecular Devices Calcium-3 Kits as the dye of choice in our current HTS campaigns.

• Investigating ligand-receptor dissociation rates using FLIPR
  Steven J. Charlton, Ph.D., Novartis Horsham Research Centre

  The kinetics of ligand dissociation from a G protein-coupled receptor (GPCR) can be an important consideration for a drug discovery programme. Few compounds, however, are examined for receptor kinetics due to the nature of the low throughput radioligand binding assays often employed. The availability of a simple, high throughput method for assessing compound off-rate would allow this property to be closely tracked alongside potency during a drug discovery programme.

  We had previously noted that in a FLIPR calcium assay, single concentrations of antagonist not only shifted the concentration-response curve of an agonist to the right, but also caused a significant reduction in the maximum response elicited, consistent with competitive, irreversible antagonism. When the same compounds were tested in radioligand binding experiments at equilibrium, however, they proved to be competitive, reversible antagonists. We hypothesised that, due to the rapid kinetics of calcium release from intracellular stores, agonist would not have sufficient time to reach equilibrium with the pre-incubated antagonist and receptor before the measured response was observed. This may result in a proportion of receptors being effectively irreversibly labelled during the short assay time, implying that the degree of depression of maximal agonist-induced response would be proportional to the dissociation rate of the antagonist. To test this hypothesis we examined a series of 30 antagonists with a broad range of off-rates (t_ 0.14- 609.4 min in a binding assay) in the FLIPR assay and ranked them based on degree of depression of the maximal response (0-62 % depression). There was a good correlation between depression of calcium response and the t_ determined using the radioligand binding assay (r² = 0.8).

  In summary, we have demonstrated that the degree of depression of the maximal agonist-induced calcium signal is directly proportional to the dissociation rate of the antagonist. This simple and rapid assay is capable of ranking antagonists based on their off-rates and represents a novel application of FLIPR.

• Functional cell-based assays in the FLIPR identify state-dependent calcium channel antagonists
  Menghang Xia, Ph.D., Department of Molecular Pharmacology, Merck & Co., Inc.

  State-dependent interactions of Ca²⁺ channel antagonists in a functional cell-based FLIPR assay, which measures Ca²⁺ influx through a voltage-dependent calcium channel, are described. The cell line used in this study was stably transfected with the L-type Ca²⁺ channel complex. It also was co-transfected with the Kir 2.3 inward rectifier K⁺ channel, which allowed for changes in cell membrane potential through alteration of extracellular [K⁺]_o. High [K⁺]_o depolarized the cells and increased the Ca²⁺ influx. The EC₅₀ for this response was 11 mM. The Ca²⁺ influx was inhibited by the L-type Ca²⁺ channel antagonist, nimodipine (IC₅₀= 59 nM), after preincubation under normal physiological [K⁺]_o conditions (polarized). Under these conditions, the membrane potential was –65 mV as measured by electrophysiology (whole cell perforated patch method). Cells were depolarized to –28 mV by elevation of [K⁺]_o. The inhibitory concentration-response relationship of Ca²⁺ channel antagonists after preincubation with increased [K⁺]_o was shifted to the left compared with that observed for cells in physiological [K⁺]_o. The IC₅₀ value of nimodipine inhibition under the depolarized condition was 3 nM. The ratios of IC₅₀ values for a range of L-type antagonists tested in the polarized compared to the depolarized cells were 4 to 20-fold. The interaction of these Ca²⁺ channel antagonists with the channel expressing cells is dependent upon the state of the channel, which is modulated by changes in membrane potential. This novel FLIPR assay is useful for evaluating state dependent inhibitory potency of a large number of compounds and can be used to identify state-dependent Ca²⁺ channel antagonists.

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electrophysiology: applications of IonWorks™ HT

• State dependent screening and Ionworks™. Assay development and implementation
  Jeff Pfohl, Ph.D., Amphora

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  The introduction of IonWorks™HT presents the possibility to reshape ion channel drug discovery, by providing unprecedented capacity of electrophysiology measurements. Amphora Discovery is applying the IonWorks HT as part of a novel discovery paradigm. To broadly implement IonWorks HT, elements of assay development have been enhanced and enabling opportunities in assay design have been explored. For any assay platform, it is important to provide data of high quality and precision. Data from optimizing a HEK cell line stably expressing voltage-dependent calcium channels for seal rate, percentage of cells expressing currents, average current size per cell, stability and DMSO tolerance will be shown. This experience not only enables our assays but also will significantly reduce assay development time. The resulting assay has been used to conduct a preliminary screen to identify compounds as well as for confirmation of results from a novel microfluidic HTS assay platform developed by Amphora that measures state dependent activity of compounds. Assay designs using IonWorks HT that will be presented include recordings of channel types from primary and freshly dissociated cells, protocol designs that reveal state dependent compound activity and multiplexing of targets (HERG and N-type Calcium Channels). These data have provided the foundation for Amphora to fully implement IonWorks HT in its multi-dimensional lead generation platform.

• Development of novel high throughput patch clamp assays for ion channel targeted drug discovery
  Laszlo Kiss, Ph.D., Merck Research Laboratories

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• Sodium channel pharmacology: a practical comparison of FLIPR, IonWorks HT and traditional whole-cell recording
  James T. Limberis, Ph.D., Pharmacia

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  Voltage-gated sodium channels are critical elements of action potential initiation and propagation in all excitable cells. Unintended drug-induced blockade of cardiac sodium channels can potentially result in serious side effects including sinoatrial block, atrioventricular block, and sudden death. Therefore, we compared two in vitro assays for the ability to identify compounds that inhibit human cardiac sodium channels stably expressed in a mammalian cell line. The two assays described are the IonWorks High Throughput (HT) from Molecular Devices and traditional whole-cell electrophysiology. The advantages and disadvantages of both assays will be discussed.

advances in secondary screening

• MTS screening of physiochemical parameters in drug discovery
  Anand Sistla, Ph.D., Sugen

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  Physiochemical properties, such as solubility and partitioning, continue to be the prevalent concern associated with combinatorial chemistry and biological activity screening. This is especially true in case of compounds aimed for oral therapy. The evaluation of these parameters at an earlier stage of drug discovery presents a unique opportunity to decrease the ‘discovery to primary candidate’ attrition.

  New technologies and strategies aimed towards higher throughput screening of physiochemical parameters have been established and are continually being refined. These include experimental and computational tools, which have increased resources and acceptance throughout discovery programs.

  Current high throughput assays are geared towards ranking and selection of compounds at early stages in the program such as ‘hit to lead’. As the project progresses into ‘lead optimization’ and beyond, there is a need to obtain information with a higher degree of veracity and without a substantial decrease in throughput. Medium throughput screening (MTS) methods aimed towards screening, ranking, and enabling formulations for discovery programs will also be presented.

screening using Molecular Devices' kinase, phosphodiesterase and cAMP assays

• A quantitative comparison of IMAP, FP, and radiometric kinase assay
  Frank Bourbonais, Ph.D., Pfizer
• High throughput screening for PDE inhibitors using IMAP®
  Y. Vivienne Marsh, Array Biopharma, Inc.

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  Phosphodiesterases (PDEs) hydrolyze the second messenger molecules cAMP and cGMP to affect a variety of physiological processes. Eleven phophodiesterase families have been identified. PDEs can be distinguished from one another by substrate specificity, tissue distribution, mode of regulation, and sensitivity to inhibitors. Collectively, the literature suggests that discovering selective inhibitors of the different PDEs may be therapeutically useful. Recently, several selective PDE inhibitors have been developed for various medical indications. Many investigators use the procedure of Thompson et al (Advances in Cyclic Nucleotide Research, 1979) for assay of PDEs. This method is not amenable for high throughput screening because it uses radiolabeled substrate and requires separation of the radiolabeled product by ion exchange columns. Over the past several years, other assay platforms, such as the Immobilized Metal Assay for Phospho-chemicals (IMAP®) and Scintillation Proximity Assay (SPA) technologies, were developed for PDE assays. We decided to use IMAP® technology to screen for inhibitors against a target PDE because it is both homogeneous and non-radioactive. This presentation will review our experience with developing, automating, and implementing a high throughput screen for PDE inhibitors using IMAP®.

• IMAP PDE assays for PDE1 to PDE5 using sf9 cell lysates
  Chengjun Deng, Ph.D., Memory Pharmaceuticals

  Cyclic nucleotide phosphodiesterases (PDEs) are attractive drug targets for a variety of human diseases. Efficient high throughput PDE enzyme assays are essential for drug discovery as well as for understanding the role of these enzymes in signal transduction pathways. Up to the present, most PDE assays are still based on radioisotopically labeled cyclic nucleotides. These assays are usually low throughput and are encumbered with large amounts of radioactive waste disposal. IMAPTM method is a fluorescence-based method that is fast and better accommodates high-throughput screening. We have expressed PDE1 through PDE5 in Bac-to-Bac Baculovirus Expression System and the cell lysates are prepared from the infected-sf9 insect cells expressing PDEs. Using small volume and dialyzed cell lysates, we analyzed the activity of PDE1 through PDE5 with the IMAPTM PDE assay. The maximal activities of each PDE ranged from 130 to 250 mP. Each PDE was inhibited by its reference inhibitors and the data were comparable to those obtained by a non-IMAP method. Considering the high precision, stable activity window (difference between the signal and the background), simple procedures and rapid plate reading that this assay achieved, the IMAPTM PDE assay could be a very useful screening technology for large-scale library compound screening.

• Miniaturization of IMAP® into 1536-well format
  James Beasley, Ph.D., Pharmocopiea

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  Two separate IMAP® assays have been developed and screening is underway against Pharmacopeia's ECLIPS (tm) combinatorial libraries encompassing more than 4 million compounds. These assays were miniaturized into 1536-well Nunc® plates with a final assay volume of 8 µL. The necessary modifications required to miniaturize these assays will be discussed. Issues related to compound interference will be presented for IMAP and compared to FP assays. Screen quality parameters for the IMAP assays such as assay reproducibility, delta mP, Z factor, and assay sensitivity will be presented.

improving the cost effectiveness and throughput of screening platforms using Analyst® and FLIPR

• Evaluation of low volume 384-well plates on FLIPR
  Theresa Truitt, Hoffman-La Roche Inc.

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  As the size of chemical compound libraries grow, it creates a clear need to control the “cost per well” of a high throughput screen. One way to reduce costs while utilizing existing FLIPR screening systems is to consider utilization of low volume 384 (LV384) well plates. A new stage, designed and installed by MDC, allows interchangeability between standard 384 MTPs and LV384 well plates. We compared the equivalence of using these two distinct plate types by assessing the performance parameters of several known GPCRs stably transfected into CHO-K1 cells. Parameters evaluated include: signal/background ratio; standard deviation of the signal across the plate; Z’ values; agonist and antagonist dose response curves, as well as the ability to run a small (<10,000) compound library screen. Comparison of MDC’s standard “No-Wash” dye to the new Calcium-3 dye was also generated. Finally, we investigated the utility of cell plating into LV384 well plates via MultiDrop™, Aquamax™, and FlexDrop™ dispensing systems. Our results suggest that LV384 FLIPR screening is a viable alternative to consider when developing GPCR based calcium flux assays.

• Using Kalypsys' uHTS technologies to expand the drug discovery pipeline
  Daniel Sipes, Kalypsis

  Kalypsys' proprietary uHTS System enables cellular and biochemical screens to be routinely carried out at a rate in excess of 1,000,000 assay wells/day. Running screens at this throughput, however, generates bottlenecks upstream and downstream of screening, in assay development and hit profiling, respectively. To address these bottlenecks, Kalypsys has developed new technologies and approaches for high-speed assay development and hit profiling. When integrated with uHTS, these approaches significantly expand the drug discovery pipeline. They also enable the generation of a Chemoprint™ profile for each compound that is used to assess structure-activity relationships, compound safety and compound specificity as a first step in the drug discovery process.

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• Development and implementation of a cell-based FLIPR assay to discover agonists of the α7 nicotinic acetylcholine receptor
  Mark Wolfe, Ph.D., Pharmacia

  The α7 nAChR is a homopentameric ligand gated ion channel that is expressed at high levels in the hippocampus. Agonists of this receptor regulate sensory gating and cognition in both animal models and human studies. These data support the rationale for using the α7 nAChR as a drug target. Initial experiments indicated that it was not possible to stably express the full length α7 nAChR in mammalian cells. Therefore, we constructed and expressed a chimeric form of the α7 nAChR that contained the N-terminal ligand binding domain of the α7 nAChR coupled to the pore-forming domain of the 5-HT3 receptor (Eisele et al 1993). We found that the α7-5HT3 chimeric receptor was stably and functionally expressed in SH-EP1 cells. Previous studies established that theses cells contained no endogenous nAChRs, thus making them good recipients for this receptor. By modifying the assay buffer we established the conditions needed to utilize a calcium flux assay to measure the functional activity of the α7-5HT3 chimeric receptor. We will present evidence that under these conditions, the functional pharmacology of the α7-5HT3 chimeric receptor accurately predicted the functional pharmacology of the full-length endogenous α7 nAChR. These data validated the α7-5HT3 chimeric receptor as a drug target and allowed us to establish a novel, high fidelity cell-based HTS assay on FLIPR that rapidly identified α7 nAChR agonists. Directed chemical synthesis utilizing the primary assays and a panel of secondary cell-based functional assays lead to the discovery of PNU-282987, which is a potent selective α7 nAChR agonist, which has long lasting activity in multiple in vitro and in-vivo models.

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