Mammalian cell lines expressing functional nematode acetylcholine receptors and use thereof for high-throughput screening assays

Abstract

The following discloses mammalian cells lines that stably express functional nematode acetylcholine receptor subunits. The resulting expression of functional ion channels has been made possible by the stable co-expression of the chaperone protein, RIC3. These cell lines are extremely useful for the high throughput screening (HTS) of compounds, to identify new candidate parasiticidal, including nematocidal, active ingredients.

Claims

1. A stable mammalian cell line that contains within its genomic DNA and stably expresses both a gene encoding a heterologous Haemonchus contortus (H. contortus) nicotinic acetylcholine receptor (nAChR) protein subunit ACR-16 and a gene encoding a homo sapiens resistance to inhibitors of cholinesterase 3 (RIC3) protein, such that the cell line expresses a functional ion channel comprising the heterologous H. contortus nAChR protein subunit ACR-16.

2. The stable cell line of claim 1, wherein the H. contortus ACR-16 protein has at least 95% identity to a sequence as set forth in SEQ ID NO: 2 or 19.

3. The stable cell line of claim 2, wherein the RIC3 protein has a polypeptide sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 4 or 27.

4. The stable cell line of claim 1, wherein the RIC3 protein has a polypeptide sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 4 or 27.

5. A vector for producing a stable mammalian cell line, comprising both a gene encoding a heterologous H. contortus nicotinic acetylcholine receptor (nAChR) protein subunit ACR-16 and a gene encoding the homo sapiens resistance to inhibitors of cholinesterase 3 (RIC3) protein.

6. The vector of claim 5, wherein the H. contortus ACR-16 gene has at least 95% identity to the sequence as set forth in SEQ ID NO: 1, and the RIC3 gene has at least 95% identity to the sequence as set forth in SEQ ID NO: 3.

7. The vector of claim 6, wherein the ACR-16 gene has the sequence as set forth in SEQ ID NO: 1 and the RIC3 gene has the sequence as set forth in SEQ ID NO: 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) A full and enabling disclosure of the present invention, including the best mode thereof, to one of ordinary skill in the art, is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, wherein:

(2) FIG. 1 shows a scheme for using the Flip-In-T-REX system;

(3) FIG. 2 shows a map of the Haemonchus contortus (Hco) ACR-16/Human RIC3 construct;

(4) FIG. 3 shows a map of the Hco ACR-16/Hco RIC3 construct;

(5) FIG. 4 shows a map of the Dim ACR-16/Dim RIC3 construct;

(6) FIG. 5 shows video-imaging results for the Hco ACR-16/hRIC3 stable cell line (each line represents one individual cell). Highlighted are calcium signal levels obtained following application of buffer, 10 M PNU120596, and 300 nM epibatidine;

(7) FIG. 6 shows video-imaging results for the Hco ACR-16/Hco RIC3 stable cell line. Highlighted are calcium signal levels obtained following application of buffer, 10 M PNU120596, and 300 nM epibatidine;

(8) FIG. 7 shows video-imaging results for the Dim ACR-16/Dim RIC3 stable cell line. Highlighted are calcium signal levels obtained following application of buffer, 10 M PNU120596, and 300 nM epibatidine;

(9) FIG. 8 shows PNU-120956 concentration-response curves fitted using a four-parameter logistic equation of the form y=[A1A2/(1+x/x0)p)+A2, where A1 is the maximum asymptote, A2 is the minimum asymptote, x0 is the XC50, and p is the Hill slope. A value of 160 nM was calculated;

(10) FIG. 9 shows concentration response curves for cells stably expressing Hco ACR-16/hRIC3 for three orthosteric reference agonists, epibatidine (6 nM), nicotine (200 nM) and acetylcholine (480 nM);

(11) FIG. 10 shows the concentration response curve for Dim ACR-16/Dim RIC3 stable cells for an orthosteric reference agonist, epibatidine (EC50 10 nM);

(12) FIG. 11 shows the response curve for another orthosteric agonist, nicotine (EC50 350 nM);

(13) FIG. 12 shows the response curve for the allosteric modulator PNU120596 (EC50 2.4 M).

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention relates to the production of mammalian cells lines, which express functional ion channels comprised of subunits from heterologous species. The disclosure further relates to use of the stable cells lines for high throughput screening (HTS) assays to identify compounds useful in modulating these receptors.

(15) In a first aspect, the present invention provides a stable cell line that stably expresses both a gene encoding a nematode ACR-16 protein and a gene encoding a RIC3 protein. The presence of the RIC3 protein may be necessary for the expression of a resulting functional heterologous ion channel, which comprises the ACR-16 protein. As used herein, ACR-16 protein is equivalent to and used interchangeably with acetylcholine receptor subunit ACR-16.

(16) In an embodiment of the first aspect, the stable cell line contains within its genomic DNA and stably expresses both a gene encoding a functional heterologous nicotinic acetylcholine receptor (nACR) subunit protein, and a gene encoding a functional resistance to inhibitors of cholinesterase 3 (RIC3) protein. As a result of the stable expression of these two genes, the cell line expresses a functional ion channel comprising the heterologous nACR protein or subunit. In a particular embodiment, the nACR protein or subunit is an ACR-16 protein, also referred to herein as an acetylcholine receptor subunit ACR-16.

(17) In another embodiment, the stable cell line is produced by transfecting a human embryonic kidney (HEK) cell, or another mammalian cell, and selecting for stable recombinant cells.

(18) In yet another embodiment, the stable cell line contains and expresses a gene encoding a nematode ACR-16 protein or subunit. The ACR-16 protein may have a sequence having at least 90% identity to a sequence as set forth in SEQ ID NO: 2, 19, 8, 20, 21, 22, 23, 24, 13 or 25, with the proviso that the ACR-16 protein forms part of the functional ion channel.

(19) When percent identity language is used herein, it is to be understood that the protein or nucleic acid having substantial identity, at either the polypeptide sequence or polynucleotide sequence level, to one of the exemplified proteins or nucleic acids, must still exhibit sufficient structural and/or functional similarly to serve a substantially equivalent function as the exemplified protein or nucleic acid. For example, an ACR-16 protein having at least 90% identity to the polypeptide sequence as set forth in SEQ ID NO: 2 means that the referenced non-identical protein must have at least 90% of the same amino acids in the same locations, relative to SEQ ID NO: 2, and it also means that the non-identical protein must serve the substantially similar function of being able to form part of a functional nACR ion channel.

(20) Similarly, implicit in statements as to polynucleotide sequence identity is the understanding that the referenced non-identical nucleic acids must have substantial functional equivalence to the exemplified nucleic acids. Here, substantial functional equivalence of nucleic acids means that they encode for cognate polypeptides having substantial functional equivalence to one another. Accordingly, an ACR-16 gene having at least 80% identity to the polynucleotide sequence as set forth in SEQ ID NO: 1 means that the referenced non-identical nucleic acid must have at least 80% of the same nucleotides in the same locations, relative to SEQ ID NO: 1, and it also means that the non-identical nucleic acid must serve the substantially similar function of coding for a protein that is able to form part of a functional ACR-16-containing nACR ion channel.

(21) In yet another embodiment of the stable cell line, the stably-expressed ACR-16 protein has the sequence as set forth in SEQ ID NO: 2, 19, 8, 20, 21, 22, 23, 24, 13 or 25.

(22) In another embodiment, the stable cell line contains and expresses a gene encoding for a RIC3 protein having at least 90% identity to the sequence as set forth in SEQ ID NO: 4, 26, 27, 28, 29, 30, 31, 6, 32, 33, 10 or 16. In an embodiment, the RIC3 protein has a polypeptide sequence having the sequence as set forth in SEQ ID NO: 4, 26, 27, 28, 29, 30, 31, 6, 32, 33, 10 or 16.

(23) In a particular embodiment, the ACR-16 and RIC3 proteins are Haemonchus contortus (H. contortus) and Homo sapiens proteins, respectively.

(24) In another particular embodiment, both the ACR-16 and the RIC3 proteins are H. contortus proteins. The ACR-16 and RIC3 proteins may also both be Dirofilaria Immitis (D. immitis) proteins.

(25) In one embodiment, the ACR-16 protein is a D. immitis protein and the RIC3 protein is a human RIC3 protein.

(26) In another embodiment, the ACR-16 protein is a D. immitis protein and the RIC3 protein is a H. contortus protein.

(27) In a second aspect, the disclosure provides a high throughput screening (HTS) method for identifying modulators of ACR-16-containing channels

(28) In an embodiment, the HTS method may comprise the general steps of:

(29) a) culturing the disclosed stable cell lines that express functional ACR-16-containing ion channels;

(30) b) exposing aliquots of the stable cells to control and experimental compounds; and

(31) c) determining which experimental compounds are able to modulate the activity of the ACR-16-containing ion channel, thereby identifying modulators of the ACR-16-containing ion channel.

(32) In one embodiment, the determining step may comprise the step of measuring a significantly greater or lesser amount of calcium influx in the experimental aliquots of cells, relative to the amount of calcium influx in the control aliquots of cells.

(33) In a particular embodiment of the method, the calcium influx may be determined to be significantly greater in the experimental cells, indicating that the modulators are agonists of the ACR-16-containing ion channel. In an embodiment, the agonists are safe and effective parasiticidal agents for administration to animals in need thereof.

(34) In a third aspect, the disclosure provides at least one vector for producing the ACR-16- and RIC3-expressing stable cell lines.

(35) In an embodiment, the vector comprises both a gene encoding a heterologous nicotinic acetylcholine receptor (nACR) protein and a gene encoding and a resistance to inhibitors of cholinesterase 3 (RIC3) protein. The vector may contain a variety of elements known to those of skill in the art. For example, it is routine practice to incorporate antibiotic resistance cassettes, promoters, enhancers, transcription terminators, origins of replication, and any other elements required for gene expression, plasmid production/replication, and selection of stable recombinant cells. Now that the inventive combinations of ACR-16 and RIC3 have been disclosed, Applicants envision that a wide range of DNA vectors, including plasmid vectors, may be employed to produce a wide range of stable cells lines, including stable HEK cells. The stable cell line of the disclosure may be produced using the reagents and techniques disclosed herein, or they may be made using any other routine methods known by those of skill in the art.

(36) In another embodiment, the vector contains a gene encoding a functional nematode ACR-16 protein and a gene encoding a functional RIC3 protein. The gene encoding the RIC3 protein may be selected from a human RIC3 gene, an H. contortus RIC3 gene, a C. elegans RIC3 gene and a D. immitis RIC3 gene. Since applicants have surprisingly found that vectors containing an Hco ACR-16 gene and a C. elegans RIC3 gene fail to produce stable cells expressing functional ACR-16-containing channels, a non-C. elegans RIC3 gene is preferred when an Hco ACR-16 gene is present in the vector.

(37) In still another embodiment of the vector, the ACR-16 gene has at least 80% identity to the sequence as set forth in SEQ ID NO: 1, 7, 11 or 12 and the RIC3 gene has at least 80% identity to the sequence as set forth in SEQ ID NO: 3, 5, 9, 14 or 15, in any combination of ACR-16 gene and RIC3 gene, with the proviso that when the vector contains an Hco ACR-16, the RIC3 gene is not a C. elegans RIC3 gene.

(38) The ACR-16 gene may also have a polynucleotide sequence that encodes a polypeptide as set forth in SEQ ID NO: 2, 19, 8, 20, 21, 22, 23, 24, 13 or 25. Alternatively, the ACR-16 gene may encode a polypeptide that is at least 90% identical to a polypeptide sequence as set forth in SEQ ID NO: 2, 19, 8, 20, 21, 22, 23, 24, 13 or 25.

(39) In another embodiment of the vector, the ACR-16 gene has the sequence as set forth in SEQ ID NO: 3, 5, 9, 14 or 15; and the RIC3 gene has the sequence as set forth in SEQ ID NO: 3, 5, 9, 14 or 15. Any combination of ACR-16 gene and RIC3 gene is envisioned, with the proviso that when the vector contains an Hco ACR-16, it is preferred that the RIC3 gene be other than a C. elegans RIC3 gene.

(40) In yet another embodiment, the RIC3 gene may encode a polypeptide as set forth in SEQ ID NO: 4, 26, 27, 28, 29, 30, 31, 6, 32, 33, 10 or 16. The RIC3 gene may also encode a polypeptide having at least 90% identity to a polypeptide as set forth in SEQ ID NO: 4, 26, 27, 28, 29, 30, 31, 6, 32, 33, 10 or 16.

(41) In a particular embodiment of the vector, the ACR-16 gene has the sequence as set forth in SEQ ID NO: 1 and the RIC3 gene has the sequence as set forth in SEQ ID NO: 3.

(42) In a fourth aspect, the disclosure provides a method for producing cells that stably express functional ACR-16-containing ion channels, which comprises the step of stably transfecting cells with both an ACR-16 gene and a RIC3 gene. In a particular embodiment, the ACR-16 gene has the sequence as set forth in SEQ ID NO: 1 and the RIC3 gene has the sequence as set forth in SEQ ID NO: 3.

(43) The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

(44) Below discloses the development of the disclosed stable cell lines, which express functional ACR-16 channels, and are useful for the high-throughput screening of compounds capable of modulating ACR-16 channel function.

Example 1Production of Mammalian Cells Expressing Functional Nematode AChR: Hco ACR-16/Human RIC3

(45) Materials & Methods.

(46) In general, the Flp-In T-Rex-293 (Human Embryonic Kidney cells) and the Flp-In T-Rex system # FITR were used to produce the stable cell lines. The vector was pCDNA5-FRT-TO_DEST and the insert sequences were Hco ACR-16 (SEQ ID NO: 1) and Human RIC3 (SEQ ID NO: 3) (depicted in FIG. 2). Cells were grown in DMEM (#31966, Invitrogen) supplemented with 10% FCS (#10500, Gibco), 15 g/ml Blasticidin (#ant-bl-1, InVivoGen) and 80 g/ml Hygromycin B (#10867, Invitrogen). Cells were grown at 37 C., 5% CO2 and 90% humidity, and passaged using Accutase (#091000449, Sigma). Finally, inductions were carried out using 0.1 g/ml doxycycline at for 24 h at 37 C.

(47) The Flp-In T-Rex expression system allows the generation of stable mammalian cell lines. The gene of interest can be integrated at a specific genomic location called Flp Recombination Target (FRT) site. The integration of the gene of interest into the genome is mediated through a Flp recombinase. With this system, the generation of stable cell line is rapid and efficient as it permits the generation of isogenic cell lines without clonal selection. This system is illustrated in FIG. 2, 19, and is available in more detail in the product manuals for pcDNA5/FRT/TO (herein incorporated by reference in their entirety. The inducible expression vector was designed for use with the Flp-In T-REx System (Cat. no. V6520-20).

(48) The Haemonchus contortus ACR-16 clone was obtained from the University of Manchester and was back-mutated to match the publicly available accession number. The following primers were then used to equip the clone with suitable restriction sites for cloning into pCDNA5dual-FRT-TO_DEST. Cloning was done using the InFusion technology. SEQ ID NO:17 (9924-01 forward primer PmlI InFusion) 5-AGG TGT CGT GAA CAC GTG CCA CCA TGT GGA GCT TGC TGA TCG C-3; SEQ ID NO:18 (9924-02 reverse primer PmlI InFusion) 5-AGC GGC CGC GAC CAC GTG CTA GGC GAC CAG ATA TGG AG-3. The human RIC3 was taken from a clone that was cloned from adrenal tissue at Sanofi. For cloning into pCDNA5 dual-FRT-TO_DEST, the Gateway cloning technology was used.

(49) Transfections. One day prior to transfection, 1.5*10.sup.6 Flp-In-T-Rex-293 or CHO cells were seeded in 10 ml DMEM or HAM-F12 containing 10% FCS into a Petri Dish (=100 mm) and incubated at 37 C./10% CO2 overnight. Using the Lipofectamine transfection reagent, cells were co-transfected with the Flp recombinase expression plasmid pOG44 and the pCDNA5 dual-FRT-TO-target with a 9:1 ratio. For the transfection of one dish, 10.8 g of pOG44 and 1.2 g of pCDNA5 dual-FRT-TO-target were mixed to 500 l Opti-MEM I medium containing 72 l Lipofectamine reagents. After 20 minutes of incubation at room temperature, the transfection reagent/DNA complex was distributed drop wise onto the cells. Flp-In-T-Rex-293 or CHO cells were incubated at 37 C./10% CO2. Five hours after transfection the cells were washed and fresh culture medium was added to the cells.

(50) Forty-eight hours after transfection the cells were washed and fresh cultivation medium containing the selection antibiotic was added. Flp-In-T-Rex-293-HcoACR-16 hRic3 cells were selected with 80 g/ml hygromycin. The culture medium was exchanged every 2-3 days until a resistant population of cells had grown. After two to three weeks of selection, the cells were cultivated in T75 flasks for scale-up and batch production.

(51) In parallel, a transitory expression of HcoACR-16 with Hco and Cel Ric3 was performed in the HEK parental cell line. The combination of Hco AChR-16+Cel Ric3 did not yield functional AChR16 channels while expression of HcoACR-16+Hco or human Ric3 did yield functional channels.

(52) Cells were harvested from the culture flasks by a short treatment (2-5 minutes) with accutase, resuspended in culture medium and centrifuged at 1000 rpm/10 min. Cells were resuspended in 90% fetal calf serum containing 10% DMSO and stored frozen in liquid nitrogen. All cell lines in culture and in the frozen stock were mycoplasma-free. DMEM (Gibco 31966) supplemented with 10% FCS (Gibco 10500) and 100 g/ml Penicillin & Streptomycin (Gibco 15140); 80 g/ml Hygromycin B (Invitrogen 10687); and 15 g/ml Blasticidin (InVivoGen ant-bl-1). Subculturing was performed by the following procedure: detach cells with Accutase (Sigma A6964), 1 ml/T75 cm2 flask Count: Vi-Cell (Beckman Coulter) Split Ratio: A subcultivation inoculum of 1:10 for 3 days maintenance and 1:15 for 4 days maintenance is recommended. Environment: 5% CO2/37 C./95% RH Culture Flasks: 75 cm.sup.2 flask (Corning 430641).

Example 2Production of Mammalian Cells Expressing Functional Nematode AChR: Hco ACR-16/Hco RIC3

(53) The methods of Example 1 were used, except that the insert sequences used were Hco ACR-16 (SEQ ID NO: 1) and Hco RIC3 (SEQ ID NO: 5) (depicted in FIG. 3).

Example 3Production of Vectors for Transient Expression of Functional Nematode AChR: Cel ACR-16 and Cel RIC3

(54) The methods of Example 1 were used, except that the vector was pCDNA3.1neo_DEST and the insert sequences were Cel ACR-16 (SEQ ID NO: 7) and Cel RIC3 (SEQ ID NO: 9). Transfections were carried out using FuGENE 6 (#E2691, Promega).

Example 4Production of Mammalian Cells Expressing Functional Nematode AChR: Dim ACR-16/Dim RIC3

(55) As in Example 1, Flp-In T-Rex-293 (Human Embryonic Kidney cells) and the Flp-In T-Rex system # FITR were used to produce the stable cell lines. The vector was pCDNA5-FRT-TO_DEST and the inserts were codon-optimized Dim ACR-16 (SEQ ID NO:12) and codon-optimized Dim RIC3 (SEQ ID NO:15) (depicted in FIG. 4). Applicants cloned Hco RIC3 from Hco cDNA prepared from field isolates, and to their knowledge, no one has previously published this sequence.

Example 5Evaluation of Mammalian Cells Expressing Functional AChR Using Calcium Mobilization Fluorescence Assays

(56) Video-Imaging Setup, Assay Protocol for Fluorescence-Ca2+ Measurement

(57) Fluorescence intensity was measured on a Hamamatsu station (camera, polychromator, Simple PCI software) with two excitation wavelengths 340 (free fura2) & 380 nm (Ca2+fura2) and emission beyond 510 nm.

(58) Working Assay Buffer=Ringer's Solution:

(59) In distilled water: to mM Hepes (Sigma H7523), 150 mM NaCl (Sigma S6191), 4 mM KCl (Sigma P5405), 2 mM CaCl.sub.2 (Sigma C4901), 1 mM MgCl.sub.2 (Sigma M2670), adjusted at pH 7.3 by NaOH 2M (Fisher). All products are in powder form except NaOH in solution form. Osmolarity was set up at 301 mmol/kg.

(60) Assay Protocol for Video-Imaging Ca2+ Measurement:

(61) Ca.sup.2+ increase was monitored using Flura-2 (AM) dye and imagery of fluorescence was performed on a Hamamatsu platform (Nikon Eclipse TE2000U+Photonics Polychromator+Orca Camera+SimplePCI software). Cells were seeded in growth medium with 1 g/ml of doxycycline onto Labtek Chamber slideck, poly-lysine coated at a cell density of 20010.sup.3 cells/chamber in 1 mL. Induction: doxycycline 1 g/ml for 24 h @ 37 C. @ 5% CO.sub.2, 1 M of Fura-2-AM was added for 20 minutes at 37 C./5% CO.sub.2 Medium was replaced by assay buffer (Ringer's solution). Fluorescence intensity was measured above 510 nm with both excitation wavelengths of 340 (free-fura2) and 380 nm excitation (Ca.sup.2+ bound fura2). Calcium increase was triggered by reference agonist application: 300 nM epibatidine in assay buffer following pre-incubation of 10 M PNU-120596.

(62) TABLE-US-00001 TABLE 1 Reagents for the Calcium Imaging Studies Materials Supplier Cat. No. Function Flura-2 AM Molecular F1221 Calcium sensitive fluorescent Probes dye HEPES SIGMA H7523 Ringer's Buffer solution NaCl SIGMA S6191 Ringer's Buffer solution KCl SIGMA P5405 Ringer's Buffer solution MgCl.sub.2 SIGMA M2670 Ringer's Buffer solution CaCl.sub.2 SIGMA C4901 Ringer's Buffer solution NaOH Fisher Ringer's Buffer solution pH adjusted Epibatidine SIGMA P178 nAChR agonist Reference PNU120596 nAChR positive allosteric modulator reference Flp-In-293-Hco ACR-16 hRic3 Cell line expressing the Hco ACR-16 and hRic3 (i.e. the line described in Example 1) Flp-In-293 Parental cell line Poly-D-Lysine SIGMA P6407 Coating Lab-Tek II Chamber slide NUNC 155379 Assay plate

(63) TABLE-US-00002 TABLE 2 Epibatidine statistics Std Event Est. N Mean Dev Median nMAD Min Max Epibatidine 300 nM Rmin 98 1.02 0.12 1.04 0.07 0.49 1.39 Epibatidine 300 nM Rmax 98 2.62 1.69 2.34 1.42 0.49 9.16 Epibatidine 300 nM Ratio 98 2.51 1.57 2.15 1.31 0.99 8.10 Epibatidine 300 nM Reaction 51 17.69 9.79 15.03 0.00 15.03 80.20 Time Epibatidine 300 nM AUC 98 271.25 141.44 253.67 110.84 33.42 869.8

(64) Assay Protocol for Ca2+ Measurement in 384-Well Plate Format on FDSS6000 (Hamamatsu)

(65) The Ca.sup.2+ increase through either Hco or Dim ACR-16 was monitored using Fluo-4AM dye and measured by a FDSS6000 platform (Hamamatsu). Twenty-four hours before the experiment, cells were seeded in growth medium into 384-well black, clear bottom poly-lysine coated plates, at a cell density of 10.000 cells/well in 50 l complemented with 1 g/ml doxycycline.

(66) Medium was replaced by washing three times with assay buffer, keeping a residual volume of 25 l per well. Subsequently 25 l of dye loading buffer were added and the plate was incubated for 1 h at RT.

(67) Dye loading buffer was removed by washing three times with assay buffer (Cell washer BioTek), keeping a residual volume of 50 l per well.

(68) Plates were transferred to the FDSS6000 reader and measured for agonist response by adding 5 l of agonist solution.

(69) TABLE-US-00003 TABLE 3 Calcium Imaging Reagents Cat. Materials Supplier No. Function Fluo-4/AM Invitrogen Calcium sensitive fluorescent dye HBSS (10x) with Invitrogen 14065 Buffer solution calcium/magnesium Flp-In-293-Hco ACR-16/ Cell line expressing hRIC3 hybrid ACR-16 Epibatidine nAChR agonist reference PNU-120596 nAChR positive allosteric modulator reference 384-well plate (poly- BD Assay plate lysine coated)

(70) TABLE-US-00004 TABLE 4 Assay Buffer Composition Reagent Chemicals Remarks Assay buffer HBSS (+Ca/+Mg) 1x 1 mM CaCl.sub.2 20 mM Hepes 0.001% Pluronic acid Set to pH 7.4 Agonist/ Assay buffer Fresh solution of compound epibatidine/nicotine was buffer prepared Dye loading Assay buffer containing: Fluo-4/AM is added from a buffer 4 M Fluo-4/AM 0.5 mM stock solution in DMSO 0.1% BSA (1 mg diluted in 910 l DMSO, protected from light)

(71) TABLE-US-00005 TABLE 5 Robustness values obtained for 384-w format assay using Hco ACR-16/hRIC3 cell line Signal amplitude with 10 M PNU120596 + 300 nM epibatidine Conditions application (RFU) Z 24 h @ 37 C. 1503 0.89 24 h @ 37 C. + 24 h @ 30 C. 2160 0.90

(72) As a measure of assay robustness, the Z value is calculated as follows using the means () and standard deviations () of both positive (p) and negative (n) controls (.sub.p, .sub.n, .sub.p and .sub.n):
Z=13*(.sub.p+.sub.n)/|.sub.p.sub.n|

(73) For a high-throughput screen, a Z value of 1 is ideal and greater than 0.5 is considered excellent. Z is typically calculated for each plate with plate-specific positive and negative controls.

(74) TABLE-US-00006 TABLE 6 EC50 values for reference compounds vs Hco ACR-16/hRIC3 cell line Reference compounds EC50 value (M) Remarks Epibatidine 0.006 Revealed by co-application of PNU-120956 Nicotine 0.20 Revealed by co-application of PNU-120956 Acetylcholine 0.48 Revealed by co-application of PNU-120956 PNU-120956 0.16 Revealed by co-application of EC100 orthosteric reference agonist

(75) TABLE-US-00007 TABLE 7 Robustness values obtained for 384-w format assay using Hco ACR-16/Hco RIC3 cell line Signal amplitude with 10 M PNU120596 + 300 nM epibatidine Conditions application (RFU) Z 24 h @ 37 C. 680 0.81 24 h @ 37 C. + 24 h @ 30 C. 1601 0.85

(76) TABLE-US-00008 TABLE 8 Hco ACR-16/Hco RIC3 stable cell line Result on FDSS6000 setup Reference compounds EC50 value (M) Remarks Epibatidine 0.005 Revealed by co-application of PNU-120956 PNU-120956 1.4 Revealed by co-application of EC100 orthosteric reference agonist

(77) TABLE-US-00009 TABLE 9 Dim ACR-16/Dim RIC3 stable cell line Results on FDSS6000 setup Reference compounds EC50 value (M) Remarks Epibatidine 0.005 Revealed by co-application of PNU-120956 Nicotine 0.4 Revealed by co-application of PNU-120956 PNU-120956 2.4 Revealed by co-application of EC100 orthosteric reference agonist

(78) TABLE-US-00010 TABLE 10 Robustness values obtained for 384-w format assay using Dim ACR-16/Dim RIC3 cell line Signal amplitude with 10 M PNU120596 + 300 nM epibatidine Conditions application (RFU) Z 24 h @ 37 C. + 24 h @ 30 C. 450 0.81

(79) The invention will now be set forth in the following non-limiting claims.