WO2002075273A2 - Dispositif et procede de triage, de diagnostic et/ou de dosage d'un agoniste et/ou antagoniste d'un recepteur couple au calcium - Google Patents

Dispositif et procede de triage, de diagnostic et/ou de dosage d'un agoniste et/ou antagoniste d'un recepteur couple au calcium Download PDF

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WO2002075273A2
WO2002075273A2 PCT/EP2002/003121 EP0203121W WO02075273A2 WO 2002075273 A2 WO2002075273 A2 WO 2002075273A2 EP 0203121 W EP0203121 W EP 0203121W WO 02075273 A2 WO02075273 A2 WO 02075273A2
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receptor
agonist
antagonist
cell
solid support
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PCT/EP2002/003121
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WO2002075273A3 (fr
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Vincent Dupriez
Marc Parmentier
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Euroscreen S.A.
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Priority to AU2002256694A priority Critical patent/AU2002256694A1/en
Publication of WO2002075273A2 publication Critical patent/WO2002075273A2/fr
Publication of WO2002075273A3 publication Critical patent/WO2002075273A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention is related to a screening, diagnostic and/or dosage method of an agonist and/or an antagonist for a membrane-linked receptor, it is also related to a corresponding device allowing the performance of said method, and it is further related to the agonist and/or antagonist of said calcium-coupled receptor identified by said method and device.
  • GPCR G-protein-coupled receptors
  • Changes in calcium concentration can be detected by several means and methods, such as the use of fluorescent dyes (for example: fura-2, fluo-3 and indo-1) .
  • Another method for intracellular calcium concentration measurement is the use of cell lines overexpressing a GPCR and apoaequorin, such as described by Sheu et al (1993).
  • cells expressing apoaequorin are incubated with coelenterazine, which is the co-factor of aequorin.
  • coelenterazine enters the cell and conjugates with apoaequorin to form aequorin, which is the active form of the enzyme.
  • intracellular calcium concentration increases.
  • aequorin for calcium measurement has limitations.
  • the classical aequorin assay light is emitted only during 20 to 30 seconds after activation of the GPCR. Thus the emitted light has to be recorded during the few seconds following agonist addition to the cells.
  • This flash-type signal is due to the fact that (1) the intracellular calcium increase triggered by GPCR is only transient, (2) as mentioned earlier, after oxidation of coelenterazine, apoaequorin must recombine with coelenterazine to be able to emit light again and (3) when aequorin is reconstituted with native coelenterazine or classical coelenterazine derivatives, it has a very rapid response to the calcium increase and emission of light follows the profile of the calcium wave.
  • measurement of an intracellular calcium increase at a high throughput requires complicated and expensive instruments; light measuring instrumentation is required to have built-in injectors so that light can be recorded directly after an addition.
  • the EP Patent Application No. 0341477 teaches the expression of the jellyfish photoprotein aequorin in a mammalian cell system, by cloning gene pAQ440 specifying the biosynthesis of the aequorin into an expression vector plasmid of a mammalian cell system, and by subjecting the resulting plasmid to transfection and producing the photoprotein aequorin in the mammalian cell.
  • the US Patent No. 5,422,266 describes a gene encoding apoaequorin protein included in a vector capable of expressing the apoaequorin in a micro-organism such as E. coli .
  • the US Patent No. 5,714,666 describes mammalian cell lines or transgenic animals expressing apoaequorin and a receptor involved in the modulation of intracellular calcium. This document also describes a method of measuring intracellular calcium comprising adding coelenterazine cofactors to said mammalian cells expressing apoaequorin and measuring photoemission where emission of photons is indicative of intracellular calcium concentration .
  • PCT Patent Application No. WO 00/02045 teaches a method in which agonists and/or antagonists for calcium-coupled receptor can be screened for using luminometers equipped with one or six injectors.
  • the maximum throughput that can be attained with said method and conventional luminometers can only be considered as "medium throughput" by today's state of the art (i.e. 10 000 data points/day) .
  • This relative intensity is calculated through the ratio of the luminescence intensity of the semi-synthetic aequorin to that of the natural aequorin.
  • the "aequorin h" has a high relative intensity and is currently used in cell-based aequorin assays.
  • the methods of the state of the art require firstly the addition of the native coelenterazine cofactor - or of a chemical derivative of coelenterazine that does not confer significantly different kinetic properties to the enzyme - to cells from a mammalian cell line expressing apoaequorin and incubation to reconstitute a functional aequorin, secondly the preparation of the agent affecting a receptor involved in the modulation of intracellular calcium concentration, thirdly the mixing of said cells with said agent, and finally the measurement of the photoemission.
  • light is emitted only during 20 to 30 seconds after activation of the GPCR. Therefore, the recording of the emitted light must be performed during the few seconds following agonist addition to the cells.
  • the present invention aims to provide a method and means, for detecting biologically active substances, especially agonists and/or antagonists for calcium-coupled receptors, said methods not presenting the drawbacks of the state of the art.
  • a main aim of the present invention is to provide such method and means which allow the detection of biologically active substances, preferably suitable for high-throughput analysis, and which could be adapted to microtiter plates without requiring the addition of built- in injectors inside the screening device.
  • Another aim of the present invention is to provide an easy and non-expensive method which could be easily automated (preferably suitable for high throughput analysis ) .
  • the present invention relates to a screening, diagnosic and/or dosage method of a biologically active substance, preferentially a known or unknown agonist (full agonist or partial agonist) and/or a known or unknown antagonist (neutral antagonist or inverse agonist) for a calcium-coupled receptor, comprising following steps:
  • An intrinsic activity is the maximal stimulatory response induced by a compound on a receptor in relation to that of a given reference compound.
  • the numerical value of said intrinsic activity can range from maximal response for full agonists to zero for antagonists, the fractional values within this range denote partial agonists .
  • An inverse agonist is a compound which acts on the same receptor as that of the agonist, yet it produces the opposite effect. Said inverse agonists are also called negative antagonists or reverse agonists.
  • the term “screening” is the search for a specific compound out of a pool of different compounds .
  • the term “diagnostic method” is an analytical method wherein the compound is characterized as being agonist or an antagonist, and preferentially indicating which type of agonist or antagonist said compound is (eg. partial agonist, full agonist, neutral antagonist or inverse agonist) .
  • the dosage method indicates the efficacy by which the compound binds to said receptor, this is preferentially indicated by EC50 or IC50 values.
  • said biological active substance is a substance of natural origin or synthesized in vi tro.
  • the present invention allows said methods to be performed in at low cost and potentially at high- throughput, this in contrast to similar prior art methods.
  • said "calcium- coupled receptor” refers to a receptor which is naturally coupled to a calcium pathway or to a receptor which naturally is not directed to a calcium pathway but is redirected to this particular pathway. It is known by a person skilled in the art that, for example certain G- protein coupled receptors may couple to Gs, Gi, Go, Gq-11 proteins. Receptors coupling naturally to a G-protein belonging to the Gq-11 family results automatically in the modulation of the intracellular calcium pathway. In contrast, other receptors regulate the production of cAMP via Gs and Gi and do naturally not influence the intracellular calcium level directly. In addition, it is known by a person skilled in the art that said latter receptors may couple promiscuously to G16 which leads to the change of intracellular calcium. Consequently, said receptors may move to another intracellular pathway depending on the coupling molecules present in the cell. It is not excluded that receptors other than GPCR may be directed to the calcium pathway.
  • the present invention illustrates that, despite their low relative intensities, said semi-synthetic aequorins emit sufficient light to enable quantitative calcium changes to be determined.
  • aequorins 6, 7, and 8 (i) have a high half-total time, which is the time required to emit 50% of the total light compared to the other aequorins in the same assay conditions. Due to the combination of a longer half- total time and the relatively high light-emitting capacity of said compounds (although with low relative intensity) present inventors found surprisingly that said aquorins are superior for measuring intracellular calcium changes using an inexpensive luminometer.
  • the present invention illustrates that aequorin i allows the formation of a delayed signal, permitting the use of low-cost luminometers and low cost assay plates without interfering with the quality and/or sensitivity of the assay as such. Therefore the present invention proposes to use coelentarazine i (or derivatives thereof such as 6 and 7) in order to reconstitute "aequorin i" in assays allowing the performance of high-throughput assays in a low cost mode.
  • aequorin is defined as a natural wild type aequorin or derivative thereof (improved aequorin comprising one or more specific mutations, or homologous proteins of aequorin which are, for instance, described in the document EP-0 341 477, US-5 , 422 , 266 , US-5,714,66, EP-0 540 064, EP-0 187 519 (US 5,541,309), Stables et al . , Analytical Biochemistry, Vol.252, p.115-126 (1997) (US 5,360,728) .
  • coelenterazine i and derivatives thereof, are defined as the molecule (s) presented in the formula as depicted in Figure 3, as well as its derivatives allowing similarly the formation of a delayed signal, preferably a light emission which could be recorded between about 5 and about 20 seconds or more after mixing of the cells and the biologically active substance ( s) .
  • Derivatives of said "coelenterazine i" could be similar to coelenterazine, comprising substitution by another halogen (F, Cl, Br) or wherein one or more non-functional groups are substituted by other non-functional groups by methods well-known by the person skilled in the art (see also crystallographic analysis of aequorin and coelenterazine (Head J.F.
  • the present invention also relates to a screening, diagnostic and/or dosage method, wherein the derivative of coelenterazine i, is a compound similar to coelenterazine i as depicted in Figure 3 wherein the I is substituted by another halogen chosen from the group consisting of F, Cl, Br; or wherein one or more non-functional groups of coelenterazine i are substituted by other non-functional groups.
  • coelenterazine i could be presented and expressed by cell(s).
  • said cells may be adhering cell(s) or cell(s) in suspension.
  • said cells may be of eukaryotic or prokaryotic origin.
  • said eukaryotic cells may be of human, mammalian, animal, plant, fungi, or of yeast origin.
  • said cells may be bacterial cells .
  • the method which is used to perform the screening, diagnostic and/or dosage method may vary depending on the compound being analysed. Certain manipulations are not mentioned in the listing of the performed steps of the methods of the present invention as they may be logically deduced from the description itself.
  • the solid support is kept outside the luminometer when not transferred to the luminometer, or the mixing is performed within the lunimometer when the solid support is already transferred to the luminometer.
  • the screening, diagnostic and/or dosage method of a known or unknown agonist or a known or unknown inverse agonist for a calcium-coupled receptor may comprise the following successive steps:
  • step (d) optionally incubating said cells with said tested substance, (e) optionally transferring said solid support to a luminometer when said solid support was not previously inserted in the luminometer in step (c) , (f) obtaining a measurement of light emitted by said cell (s) , (g) determining from the results of said measurement whether the tested substance is an agonist or an inverse agonist of said receptor, and, possibly determining the affinity of said tested substance for said receptor, and,
  • step (h) possibly recovering said agonist or said inverse agonist of said receptor from the reaction mixture of step (d) .
  • this basal calcium level may consume the activated aequorin resulting in the production of basal levels of light.
  • Inverse agonists can be easily identified through the detection of the lowering of said basal level upon treatment of said cells with said inverse agonist.
  • a reference molecule is used in the experiment (preferentially a full agonist) allowing the comparison of the intensity of the emitted light for both compounds when incubated with the cells of the present invention .
  • the screening, diagnostic and/or dosage method for a known or an unknown antagonist (neutral antagonist or inverse agonist) according to the present invention may comprise the following successive steps :
  • step (h) determining from the results of steps (e) and/or (g) whether the tested substance is an antagonist of said receptor, and, possibly determining the affinity of said tested substance for said receptor, and,
  • said receptor may become occupied by the antagonist before the agonist has the possibility to bind and activate the receptor.
  • the activity of said antagonist is thus studied by their capacity for blocking the agonist activation of said receptor.
  • said inhibition is reflected by the inhibition of the light emission.
  • the screening, diagnostic and/or dosage method for a known or an unknown antagonist (neutral antagonist or inverse agonist) according to the present invention may comprise the following successive steps: (a) contacting a known agonist of said calcium-coupled receptor with a solid support,
  • step (g) optionally transferring said solid support to a luminometer and possibly obtaining a second measurement of light emitted by said cell(s), (h) determining from the results of steps (e) and/or (g) whether the tested substance is an antagonist of said receptor, and, possibly determining the affinity of said tested substance for said receptor, and, (i) possibly recovering said antagonist of said receptor from the reaction mixture of step (f).
  • the screening, diagnostic and/or dosage method for a known or unknown agonist or a known or unknown inverse agonist according to the present invention may comprise the following successive steps:
  • step (b) mixing said agonist or inverse agonist (tested substance) with the cell(s) of step (a) in a vial other than the solid support,
  • step (c) adding the mixture obtained in step (b) on a solid support ,
  • step (g) possibly recovering said agonist or said inverse agonist of said receptor from the reaction mixture of step (d) .
  • the screening, diagnostic and/or dosage method of a known or an unknown agonist (full or partial) or a known or unknown inverse agonist according to the present invention may comprise the following successive steps : (a) contacting one or more cells with a solid support, said cell(s) expressing apoaequorin and said calcium-coupled receptor, incubating said cells with coelenterazine i as depicted in Figure 3 or a derivative thereof in order to reconstitute active aequorin by said cell(s),
  • the screening, diagnostic and/or dosage method of known or an unknown antagonists (neutral antagonist or inverse agonist) for a calcium- coupled receptor may comprising the following successive steps: (a) contacting one or more cells with a solid support, said cell(s) expressing apoaequorin and said calcium-coupled receptor, incubating said cells with coelenterazine i as depicted in Figure 3 or a derivative thereof in order to reconstitute active aequorin by said cell(s),
  • step (f) optionally moving said solid support in a luminometer if not inserted at step (d)
  • step (g) possibly obtaining a second measurement of an emitted light by said cell(s), (h) determining from the results of step (d) and/or (g) whether the tested substance is an antagonist of said receptor, and, possibly determining the affinity of said tested substance for said receptor, and, (i) possibly recovering said antagonist of said receptor from the reaction mixture of step (e) .
  • the screening, diagnostic and/or dosage method of a known or an unknown antagonist (neutral antagonist or inverse agonist) for a calcium- coupled receptor comprising the following successive steps:
  • step (c) transferring the cells of step (b) to a solid support
  • step (i) possibly recovering said antaagonist of said receptor from the reaction mixture of step (f ) .
  • the screening, diagnostic and/or dosage method for a known or a unknown antagonist may comprise the following successive steps:
  • step (f) determining from the results of said measurement whether the tested substance is an antagonist of said receptor, and, (g) possibly recovering said antagonist of said receptor from the reaction mixture of step (d) .
  • an agonist and an antagonist for the same receptor is added at the same time to the cells expressing said receptor. If in this case the agonist binds before the antagonist onto this receptor, the receptor may become activated resulting in the emission of light. Depending on the binding kinetics of the compound onto said receptor, said agonist may dissociate from said receptor and be replaced by the antagonist. In this case, antagonistic properties may be detected using the method according to the present invention.
  • the screening, diagnostic and/or dosage method of a known or an unknown antagonist (neutral antagonist or an inverse agonist) according to the present invention may comprise the following successive steps:
  • step (g) possibly recovering said antagonist of said receptor from the reaction mixture of step (c). More particularly, the screening, diagnostic and/or dosage method according to the present invention, characterised in that the cell expresses apoaequorin in the cytoplasm or in the mitochondria or in any other part of the cell.
  • said cell expressing the calcium-coupled receptor may also expresses proteins (endogenous or overexpressed) ensuring a coupling of said receptor (endogenous or overexpressed) to a calcium pathway.
  • said protein may be selected from the group consisting of natural G ⁇ l ⁇ protein, chimeric G-protein resulting from a fusion between two different G-proteins or phospholipase C ⁇ 2 protein or any other coupling protein or chemical.
  • the calcium-coupled receptor of the present invention may be a G-protein- coupled receptor (GPCR) .
  • the measurement of the emitted light is obtained with one or more luminometer (s) equipped with several dispensers and measurement heads or with one or more luminometer (s) wherein no built-in dispensers are present. )
  • said luminometer may be chosen from the group consisting of the NorthStar (PE- Biosystems) Microlumat (Berthold) , FLIPR (Molecular Devices) and ImageTrack (Packard).
  • the solid support is a microtiter plate.
  • the solid support is a microtiter plate, preferably a 96-wells, 384-wells, a 1536 wells microtiter plate or smaller format.
  • the present invention relates to a high throughput screening, diagnostic and/or dosage method .
  • the present invention is also related to a (preferably high-throughput ) screening, diagnostic and/or dosage device intended for the screening method according to the invention, said device comprising the following elements :
  • microtiter plate preferably a 96-well or 384-well microtiter plate
  • - means such as one or more luminometer (s) equipped with one or several measurement heads and placed beside one or several dispensers
  • the device and dispensers according to the invention comprise means for automatically performing the successive steps of the screening, diagnostic and/or dosage method according to the invention.
  • the present invention also relates to a kit for high throughput screening, diagnostic and/or dosage method of a known or unknown agonist and/or a known or an unknown antagonist of a calcium-coupled receptor using a luminometer with a several dispensers and measurement heads or using a luminometer without built-in dispensers; comprising coelentrazine i as depicted in Figure 3 or a derivative thereof.
  • the present invention also relates to unknown agonist or unknown antagonist of a receptor identified by the method according to a method of the present invention. More in particular, the present invention relates to the use of an unknown agonist and/or an unknown antagonist of a receptor in the preparation of a medicament for treating diseases influenced by said receptor.
  • Said disease may be any disease influenced by a receptor, for example this disease may be a mood-related disease, mental-related disorders or a gastrointestinal- related disease. Other diseases influenced by receptors are known by a person skilled in the art.
  • the present invention also involves the use of a method according to the present invention for the screening, diagnosis and/or dosage of a known or unknown agonist (full agonist or partial agonist) and/or a known or unknown antagonist (neutral antagonist or inverse agonist) for a membrane-linked receptor.
  • Said membrane-linked receptor may be a G-protein-coupled receptor.
  • a last aspect of the present invention is related to a method to screen for, to diagnose and/or dose a known or unknown agonist (full agonist or partial agonist) and/or a known or unknown antagonist (neutral antagonist or inverse agonist) for a membrane-linked receptor capable of modifying the cellular metabolism in a subject.
  • Said subject may be of human, animal or plant origin.
  • a last aspect of the invention is related to the unknown agonist and/or antagonist of a calcium-coupled receptor identified by the method and device according to the invention.
  • Figure 1 describes the general principle of reconstituting an active aequorin from the protein apoaequorin expressed from a plasmid transfected in a cell .
  • the coding region of said aequorin may also be present chromosomally .
  • the coelenterazine may be natural or made synthetically.
  • Figure 2 describes the principle of the cell- based aequorin assay, as in the method of the present invention.
  • the receptor is a GPCR, but it could also be any other type of receptor, such as ion-channels or tyrosine kinase receptors.
  • Figure 3 presents the structure of the natural substrate of the enzyme (Native coelenterazine) , of a derivative obtained by chemical synthesis that is currently used in cell-based aequorin assays (coelenterazine h) and of coelenterazine i as used in the method according to the invention.
  • Figure 4 presents the enzymatic properties of aequorin when it is reconstituted with different kinds of synthetic coelenterazines in an in vi tro aequorin assay, as disclosed in the literature (Shimomura et al . , 1989, Shimomura, 1991) .
  • Figure 5 gives the intensity of the emitted light (in Relative Light Units (RLU) ) recorded for 30 seconds after injecting a cell suspension into wells of a microtiter plate, said cell suspension comprising cells co- expressing apoaequorin and the serotonin receptor 5HT2B and being incubated with coelenterazine h (upper panel) or coelenterazine i (lower panel), each well containing initially either digitonin (marked as "Digi”) or serotonin
  • RLU Relative Light Units
  • Figure 6 is a graphical analysis of the delay in the luminescent signal obtained with coelenterazine i, in comparison with some other kinds of coelenterazines (h, fcp and ip) in cell-based aequorin assays.
  • Cells were treated as described in Figure 5 and their response to Digitonin (open symbols) or their response to serotonin (5HT, closed symbols)- an agonist of a receptor expressed by the cells- was analysed.
  • Figure 8 represents the results obtained with the same cell-based aequorin assay and analysis as described in Figure 7, but here cells expressed a histamine HI receptor instead of a 5HT-2B receptor, and histamine was used as an agonist of this receptor.
  • Figure 9 represents the results obtained with the same cell-based aequorin assay as described in Figures 7 and 8, but here cells expressed the orexin receptor 2 instead of a serotonin receptor, and orexin B was used as an agonist of this receptor.
  • Figure 10 gives the dose-response curve for MCH on the MCH receptor in cell-based aequorin assays using a luminometer where there is a delay between the last injection and the beginning of the recording of the emitted light. Values (RLU, Relative Luminescence Units) are plotted against the logarithm of the agonist concentration to yield the sigmoidal dose-response curves.
  • RLU Relative Luminescence Units
  • Figure 11 gives the dose-response curve for serotonin on the 5HT-2B receptor preincubated with the antagonist mesulergine in cell-based aequorin assays using either coelenterazine h or coelenterazine i. Values (RLU, Relative Luminescence Units) were plotted against the logarithm of the antagonist concentration to yield the sigmoidal dose-response curves shown. Half maximal agonist concentration (IC50) was calculated from these curves. Digitonin was used as a control of the aequorin content of the cells .
  • RLU Relative Luminescence Units
  • the method according to the invention is related to the detection of agonistic or antagonistic activities of substances of membrane-linked receptors by means of mammalian cells lines expressing apoaequorin and a said receptor (eg. GPCR) .
  • Figure 1 summarises the general principle of the calcium-dependent generation of light by aequorin following oxidation of coelenterazine, as used in the method of the present invention, while Figure 2 explains how, in said method, the binding of an agonist to a GPCR leads, through molecular signalling, to light emission.
  • Figure 3 gives the chemical structure of coelenterazine i.
  • the synthesis and chemical structure of said coelenterazine i have been previously described by Shimomura et al . (1989). This structure is compared to the structure of the natural substrate of the enzyme (Native coelenterazine) and to the structure of a derivative obtained by chemical synthesis, which is currently used in cell-based aequorin assays and which is called "coelenterazine h" .
  • the circles indicate the differences of composition between these 3 molecules.
  • coelenterazine i the hydroxyl group of native coelenterazine (-0H) has been replaced by an iodine atom (-1) .
  • GPCR requires the measurement of emitted light to be performed just after placing the cells -pretreated with or without an antagonist- in contact with the supposed agonist. This emitted light can easily be measured at low throughput using a single-tube luminometer. However, up to now, this biological system could not be used at a high-throughput scale. Indeed :
  • Luminometers able to inject simultaneously a different compound in each of the wells of a 96 or 384-well plate and start immediately the measurement of the luminescent signal are just becoming to be available on the market (for example the FDSS, made by Hamamatsu Photonics, Japan), but these devices are expensive and require the use of expensive clear-bottom plates .
  • Other luminometers are able to read all the 96 or 384-well plate at once, but are not equipped with built-in dispensers
  • the present invention provides a method for performing high-throughput screening of drugs binding to a calcium-coupled receptor (eg. GPCR) by the use of mammalian cell lines expressing apoaequorin and a calcium-coupled receptor (eg. GPCR) and by the use of a luminometer that is equipped with no or few built-in dispensers. Following this method, the solutions to be tested for (ant) agonistic activities are placed in the wells of a 96-well plate.
  • a calcium-coupled receptor eg. GPCR
  • GPCR GPCR
  • coelenterazine i to reconstitute active aequorin.
  • said cells may be cultured in suspension, if they are suitable for use in such conditions .
  • these cells are then maintained in suspension with a magnetic stirrer and the cell suspension is injected into the solutions of supposed agonist to be tested.
  • the plate is then transferred to the luminometer, at the position where measurement of the emitted light can occur.
  • Light emission is then recorded for 1 (alternatively up to 60) second(s) .
  • This method delays the emission of light by aequorin present in the cell and allows up to 384 measurements of agonist-induced aequorin light emission to be taken in 1 minute or less, using a luminometer having few or no built-in dispenser (for example with the CLIPR or the NorthStar) , when said dispensers is located at a position which is not the reading position (for example placed beside the luminometer) .
  • This method thus allows the high-throughput screening (>100 000 samples/day) of mammalian cell lines expressing apoaequorin and a calcium-coupled receptor (eg. GPCR) by the use of a luminometer devoid of built-in dispenser. This opens a new field of use for this kind of luminometer.
  • the method according to the invention is suitable for performing high-throughput analysis of calcium-coupled receptor (eg. GPCR) stimulation by known or supposed agonists or antagonist by means of cells expressing the receptor and apoaequorin.
  • GPCR calcium-coupled receptor
  • These cells may express apoaequorin in the cytoplasm, as described by Sheu et al . (1993) or Button and Brownstein (1993) or may express apoaequorin in the mitochondria, by means of the addition of a mitochondrial targeting sequence to the aequorin, as used by Stables et al .
  • proteins intended to ensure coupling of the over-expressed receptor to the calcium pathway may be the natural G ⁇ l6 protein (Milligan et al., 1996) or its murine counterpart G ⁇ l5, chimeric G proteins resulting from a fusion between two different G proteins (Komatsuzaki et al . , 1997), phospholipase C- ⁇ 2 (Park et al . , 1992), or any other "universal coupling" protein. Examples
  • a CHO cell line expressing the serotonin 5HT2B receptor, the G ⁇ l6 coupling protein and apoaequorin was established.
  • Cells were cultivated as a monolayer in HAM'sF12 medium containing 10% Foetal bovine serum (FBS) .
  • FBS Foetal bovine serum
  • the culture medium was removed and cells were incubated for 5 min at room temperature in PBS-EDTA (phosphate buffered saline solution without calcium, added with 5 mM EDTA) .
  • PBS-EDTA phosphate buffered saline solution without calcium, added with 5 mM EDTA
  • a magnetic stirring bar was added to the suspension and a magnetic stirrer was used at low speed (1 to 5 rounds per second) to maintain the cells in an homogenous suspension.
  • the magnetic stirring bar was equipped with a ring to reduce the possibility of fracturing the cells and to reduce the subsequently release of aequorin in the culture medium.
  • a culture vessel equipped for culture of cells in suspension may be used.
  • the use of the EG&G Wallac's MicroLumat-Plus microplate luminometer allows injection and direct subsequent recording of the light emissions from each well of a 96-well plate with time, enabling kinetic measurements to be made.
  • the end of the entrance tube of the dispenser was placed at the bottom of the cell suspension and the dispenser was filled with a volume of suspension 3 times the dead volume of the apparatus so that the volume occupied by the tube and pumps were completely filled with cell suspension.
  • the 96-well plate containing the solutions of agonists was then inserted into the luminometer. Then, for each well, 50 ⁇ l of the cell suspension (i.e. 25 000 cells) was dispensed into the well and the emitted light was immediately recorded during 30 seconds. After reading the first well, cells were injected into the next well and emitted light was recorded, etc. For each plate, a series of curves representing the intensity of the emitted light as a function of time for each well was displayed.
  • Results of Figure 5 show that the use of aequorin reconstituted with coelenterazine i allows the delay of the luminescent response of aequorin to the intracellular calcium concentration increase.
  • the intensity of the emitted light was integrated over 30 seconds, or from second 8 to second 30 using the Winglow software provided with the luminometer, yielding, for each well, one value representative of the emitted light and hence of the stimulation of the 5HT2B receptor by the agonist present in the well.
  • These values can be plotted against the logarithm of the ligand concentration to generate dose-response curve as shown in Figure 7. These allow the determination of half-maximal response doses (EC50) for each ligand.
  • Figure 7 represents the dose- response curve for serotonin on the 5HT-2B receptor which represent RLU (integration of emitted light for 30 seconds for left panel or for 22 seconds for right panel) according to the logarithm of the final concentration of the serotonin.
  • RLU integrated of emitted light for 30 seconds for left panel or for 22 seconds for right panel
  • the said integrated measurement was then plotted against the logarithm of the concentration of the agonist, to yield a typical sigmoidal dose-response curve for cells loaded with coelenterazine h or with coelenterazine i.
  • the light emitted during the first 8 seconds after the mixing was omitted, and integration of the signal was performed between second 8 and second 30.
  • FIG. 8 represents the same experiment and analysis as described in Figure 7, but here cells expressed the histamine HI receptor instead of a serotonin receptor, and histamine was used as an agonist of this receptor instead of serotonin.
  • Figure 8 shows that again, the use of coelenterazine i instead of coelenterazine h allows the signal that occurs between the mixing of the cells with the agonist and the start of the light measurement to be excluded while keeping good relevance of the recorded data.
  • a CHO cell line expressing the orexin receptor 2, the G ⁇ l ⁇ coupling protein and apoaequorin was established. Cells were treated as described in Example 1 and were dispensed after dilution (50 ⁇ l/well, corresponding to 25 000 cells) into 50 ⁇ l of solutions of known agonists for this receptor. The emitted light was recorded for a 30 second time period for each well. Dose- response curves obtained for orexin B, an agonist of this receptor, are shown in Figure 9.
  • Figure 9 represents the same experiment as described in Figures 7 and 8, but here cells expressed a orexin receptor 2 instead of a serotonin receptor or a histamine receptor, and orexin B was used as an agonist of this receptor, instead of serotonin or histamine.
  • coelenterazine i instead of coelenterazine h allows the signal that occurs between the mixing of the cells with the agonist and the start of the light measurement to be excluded while keeping good relevance of the recorded data.
  • CHO-apoaequorin-MCH-R cells were prepared as described in previous experiments and 5 ⁇ M coelenterazine i were added to the cell suspension to reconstitute active aequorin.
  • the assay plate containing agonists was placed in the « NorthStar » luminometer (PE Biosysterns) and cells (20 ⁇ l/well) were injected into each well of the assay plate by means of the NorthStar dispensing system. The plate was then carried by the NorthStar device at the reading position (i.e . under the CCD camera) and measurement of the emitted light was performed for 90 seconds. With the NorthStar, delay between dispensing of the cells on the agonists and start of the measurement of the emitted light is about 16 seconds. Values (RLU, Relative Luminescence Units) were plotted against the logarithm of the agonist concentration to yield the sigmoidal dose-response curves shown in Figure 10.
  • RLU Relative Luminescence Units
  • CHO-aequorin-serotonin 2B receptor cells were prepared as described in previous examples and 5 ⁇ M coelenterazine i or coelenterazine h were added to the cell suspension to reconstitute active aequorin.
  • Cells were dispensed on the dilutions of antagonist and incubated for 30 minutes with the antagonists .
  • Serotonin a reference agonist
  • IC50 coelenterazine i
  • Digitonin was used as a control of the aequorin content of the cells.
  • This example illustrates that coelenterazine i can also be used in assays designed to detect antagonists of calcium-coupled receptors.
  • Half maximal inhibitory effect concentrations for antagonists (IC50) were the same when coelenterazine h or coelenterazine i was used (thus coelenterazine i is a valuable method for such assays) . It allows an incorporation of a delay period between the assay setup and the recording of the final signal.

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Abstract

La présente invention concerne un procédé de triage, de diagnostic et/ou de dosage d'un agoniste connu ou inconnu (agoniste entier ou partiel) et/ou d'un antagoniste connu ou inconnu (antagoniste neutre ou agoniste inverse) d'un récepteur couplé au calcium. Ce procédé consiste : - à incuber au moins une cellule exprimant l'apoaéquorine et ce récepteur couplé au calcium avec de la 'coelenterazine i' ayant la formule décrite à la figure 3 ou un dérivé de celle-ci, en vue de reconstituer l'aéquorine active par ces cellules ; - à mettre en contact cette/ces cellule(s) avec l'agoniste et/ou l'antagoniste de ce récepteur sur un support solide, et à mesurer la lumière émise par cette/ces cellule(s), ce qui permet le triage, le diagnostic et/ou le dosage de l'agoniste et/ou l'antagoniste de ce récepteur et, éventuellement à récupérer l'agoniste ou l'antagoniste de ce récepteur à partir du mélange réactionnel. La présente invention concerne également un dispositif permettant la mise en oeuvre de ce procédé, ainsi que l'agoniste et/ou l'antagoniste de ce récepteur couplé au calcium identifié à l'aide de ce procédé et de ce dispositif.
PCT/EP2002/003121 2001-03-20 2002-03-20 Dispositif et procede de triage, de diagnostic et/ou de dosage d'un agoniste et/ou antagoniste d'un recepteur couple au calcium WO2002075273A2 (fr)

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WO2004050905A1 (fr) * 2002-12-03 2004-06-17 Lutess Limited Biodetecteur eucaryote mettant en application un enzyme luminescent regule par calcium
EP1867994A2 (fr) * 2006-06-13 2007-12-19 Euroscreen S.A. Ligand pour protéine G couplé au récepteur GPR72 et utilisations correspondantes
EP2036986A1 (fr) * 2006-05-26 2009-03-18 Noacell Science Co., Ltd. Méthode de dosage continu pour mesurer la réponse au calcium médiée par les canaux ioniques et les récepteurs et l'expression ultérieure d'un gène
US7824866B2 (en) 2006-06-13 2010-11-02 Euroscreen S.A. Ligand for G-protein coupled receptor GPR72 and uses thereof

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SHIMOMURA O ET AL: "THE RELATIVE RATE OF AEQUORIN REGENERATION FROM APOAEQUORIN AND COELENTERAZINE ANALOGUES" BIOCHEMICAL JOURNAL, PORTLAND PRESS, LONDON, GB, vol. 296, no. 3, 15 December 1993 (1993-12-15), pages 549-551, XP001011593 ISSN: 0264-6021 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050905A1 (fr) * 2002-12-03 2004-06-17 Lutess Limited Biodetecteur eucaryote mettant en application un enzyme luminescent regule par calcium
EP2036986A1 (fr) * 2006-05-26 2009-03-18 Noacell Science Co., Ltd. Méthode de dosage continu pour mesurer la réponse au calcium médiée par les canaux ioniques et les récepteurs et l'expression ultérieure d'un gène
EP2036986A4 (fr) * 2006-05-26 2010-01-20 Noacell Science Co Ltd Méthode de dosage continu pour mesurer la réponse au calcium médiée par les canaux ioniques et les récepteurs et l'expression ultérieure d'un gène
EP1867994A2 (fr) * 2006-06-13 2007-12-19 Euroscreen S.A. Ligand pour protéine G couplé au récepteur GPR72 et utilisations correspondantes
EP1867994A3 (fr) * 2006-06-13 2008-04-02 Euroscreen S.A. Ligand pour protéine G couplé au récepteur GPR72 et utilisations correspondantes
US7824866B2 (en) 2006-06-13 2010-11-02 Euroscreen S.A. Ligand for G-protein coupled receptor GPR72 and uses thereof

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