WO2017109709A2 - Méthode de dosage à haut débit pour identifier des modulateurs du récepteur de nmda allostériques - Google Patents

Méthode de dosage à haut débit pour identifier des modulateurs du récepteur de nmda allostériques Download PDF

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WO2017109709A2
WO2017109709A2 PCT/IB2016/057843 IB2016057843W WO2017109709A2 WO 2017109709 A2 WO2017109709 A2 WO 2017109709A2 IB 2016057843 W IB2016057843 W IB 2016057843W WO 2017109709 A2 WO2017109709 A2 WO 2017109709A2
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nmdar
mammalian cell
calcium
cell
ligand
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WO2017109709A3 (fr
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Anke Bill
Luiz Miguel Quinn CAMARGO
Mary Ellen Digan
Hongqiu GUO
Hao-Ran RAO
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Novartis Ag
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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
    • G01N33/5008—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
    • 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
    • G01N33/5035—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 on sub-cellular localization

Definitions

  • the invention relates generally to in vitro processes that test organic co for binding to a cell membrane bound receptor.
  • NMDARs N-methyl-D-aspartate-receptors
  • NMDA receptors ionotropic glutamate receptors that require binding of two ligands, i.e., glutamate and co-factors glycine or D-serine for activity.
  • NMDARs are complex, being composed of different subtypes with distinctive properties. See, Paoletti et al., Nature Rev. Neurosci. 14: 383- 400 (2013).
  • NMDARs Most marketed drugs that target NMDARs are antagonists that block the pathological effects of having an overly activated receptor. Enhancing the activity of NMDARs can result in excitotoxicity and unwanted adverse events. Chen et al., J.
  • NMDA channel blocker memantine is administered for the treatment of Alzheimer's disease.
  • the proposed mechanism is that memantine blocks ⁇ 42 oligomers, the toxic agents in Alzheimer's disease, from over-exciting NMDARs. Cacabelos et al., Int. J. Geriat. Psychiatry 14: 3-47 (1999).
  • Schizophrenia is a chronic, severe and disabling neuropsychiatric disease with an estimated prevalence of 1 % in the general population. Negative and cognitive symptoms represent a great unmet medical need in SZ, since the impact of
  • antipsychotics current standard of care
  • Experimental evidence supports an involvement of NMDAR in the etiology and pathophysiology of schizophrenia. Normalization of NMDAR activity in the brain of patients suffering from schizophrenia is predicted to mitigate symptoms and to address the unmet medical need in this disease.
  • the limitations of currently available models include: (a) the high expression levels of functional NR2 subunit are cytotoxic, leading to cell death; (b) recombinant gene expression leads to a random ratio of subunits; (c) the need to use ketamine or similar strong inhibitors for protection against cytotoxicity leads to other disadvantages; and (d) cells are cultured in presence of ketamine to protect from excitotoxicity are not sensitive to glycine/D-serine and have limited sensitivity to glutamine in plate based assay; and (e) high levels of glycine are both present in cell media and secreted by cultured cells upon their death.
  • NMDAR channel blocker compounds are difficult to wash off the cells being tested, are themselves toxic to the cells and may confound measurements of calcium signaling in a high-throughput fashion.
  • endogenous levels of glycine and glutamate are released by the cells in culture, which then occupy the ligand binding sites and maintain the NMDAR in an activated or desensitized state.
  • Bettini's assay used ketamine in the culture medium and the cells were washed to remove ketamine. Under Bettini's conditions NMDAR showed significant constitutive activity and little sensitivity to ligand stimulation.
  • Hansen's assay was not sensitive to glycine.
  • the invention provides a method for identifying a modulator of N-methyl-D- aspartate receptor (NMDAR) function.
  • the method includes the steps of (a) transiently expressing an NMDAR protein or protein subunit on the surface of a mammalian cell; (b) culturing the mammalian cell in a culture medium, where the culture medium (i) contains an NMDAR ligand binding site antagonist; and (ii) does not contain an NMDAR channel blocker; replacing (i) the culture medium that contains the NMDAR ligand binding site antagonist and does not contain an NMDAR channel blocker with (ii) a buffer or culture medium that does not contain an NMDAR channel blocker; (c) manipulating the mammalian cell to respond in a measurable way to changes in calcium concentration in the mammalian cell; (d) while retaining the mammalian cell, (e) measuring the calcium response by the mammalian cell; (f) contacting the mammalian cell with
  • a change in calcium response by the mammalian cell identifies the test agent as a modulator of NMDAR function.
  • the buffer or culture medium does not contain an NMDAR channel blocker or any other NMDAR inhibitor other than the test agent, when the test agent is an antagonist.
  • transiently expressing NMDAR protein or protein subunit on the surface of the mammalian cell is accomplished by a transient transfection of the mammalian cell with a vector containing an NMDAR gene, and subsequent expression of an NMDAR gene.
  • the vector is a vector containing one or multiple NMDAR genes under the control of a constitutively active mammalian promotor leading to transient expression of NMDAR.
  • the vector is a baculovirus vector containing an NMDAR gene, such as a BacMam vector containing an NMDAR gene, such as a human NMDAR.
  • the transiently expressing NMDAR protein or protein subunit on the surface of the mammalian cell is accomplished by a stable transfection or a lentiviral infection of the mammalian cell with one or more vectors comprising one or more NMDAR genes, where one or more of the NMDAR genes can be inducible, such as by doxycycline.
  • the recombinant baculovirus vector containing an NMDAR gene contains one or more nucleotide sequences of a human NMDAR NR1 subunit and/or NR2 subunit together with additional sequences capable of directing the synthesis of the human NMDAR subunit in cultures of transiently transfected mammalian cells.
  • the transiently expressed NMDAR is selected from the group consisting of NR1/2A and NR1/2B and NR1/2D.
  • the NMDAR subunit is selected from the group consisting of NR1 , NR2A, NR2B, and NR2D (mouse or human sequence variants).
  • the transiently expressed NMDAR is a human NMDAR or a mouse NMDAR.
  • the mammalian cell is a human cell, such as a HEK293 cell or a human induced pluripotent stem cell (iPSC).
  • the cells in the assay are infected with BacMam virus encoding NMDAR genes.
  • the cells in the assay are protected from NMDAR-mediated excito toxicity by the addition of NMDAR inhibitor into the media during the culture of the cells.
  • the NMDAR ligand binding site antagonist binds to the glycine binding site of the NMDAR.
  • the NMDAR ligand binding site antagonist binds to the glutamate binding site of the NMDAR.
  • the NMDAR ligand binding site antagonist used in the culturing step of the invention is MDL105, 519 or CGP070667.
  • the NMDAR channel blocker not used in the method of the invention can be, for example, ketamine, memantine or MK801 .
  • manipulating the mammalian cell so that it responds in a measurable way to changes in calcium concentration in the mammalian cell is accomplished by loading the mammalian cell with a cell-permeable calcium indicator, such as a fluorescent calcium indicator.
  • a cell-permeable calcium indicator such as a fluorescent calcium indicator.
  • the fluorescent calcium indicator is calcium 6 dye.
  • manipulating the mammalian cell so that it is responds in a measurable way to changes in calcium concentration in the mammalian cell is accomplished by stably expressing a calcium sensor protein in the mammalian cell.
  • the calcium sensor protein is stably expressed in the mammalian cell by expression from a lentivirus vector comprising a calcium sensor protein gene, in a twentieth embodiment, the calcium sensor protein is CGaMP6; in a twenty-first embodiment, CGaMP6 with two aspartic acid (D) -> cysteine (C) point mutations (dCys-GCAMP6) (SEQ ID NO: 1); and in a twenty-second embodiment, CGaMP3 (SEQ ID NO: 2).
  • the measuring of the calcium response by the mammalian cell is in a high-throughput screen.
  • a high-throughput, calcium flux assay of the invention has been developed as a screening assay.
  • the assay shows sensitivity to both endogenous NMDAR co-agonists, enabling measurements of NMDAR activity in the presence of limiting amounts of either ligand. Importantly the readouts obtained from this assay exhibit expected NMDAR pharmacology.
  • the NMDAR ligand site binder binds to the NMDAR glycine binding site.
  • the NMDAR ligand site binder binds to the NMDAR glutamate binding site.
  • one or more NMDAR ligand site binder is selected from the group consisting of glycine, L-glutamate, CGP070667, AAM077, 7-CTKA, MDL105,519, L701 ,324, CGP039653 and CPP.
  • the modulator of NMDAR function is an NMDAR positive allosteric modulator, as detected by an increase in calcium response.
  • the modulator of NMDAR function is an NMDAR negative inhibitor, as detected by a decrease in calcium response.
  • the modulator of NMDAR function can bind to the NMDAR glycine binding site or to the NMDAR glutamate binding site.
  • the modulator can bind to any other site in NMDAR (by an allosteric mechanism).
  • the invention also provides a mammalian cell having a high level of an NMDAR protein on the cell surface.
  • the mammalian cell comprises a vector containing an expressible NMDAR gene, where (i) the vector is a baculovirus vector; or (ii) the vector is integrated in the mammalian cell genome, and measurable by resistance to a selection marker encoded by the vector; or (iii) the vector does not contain a selection marker and is only transiently expressed; the expressible NMDAR gene is expressed in the mammalian cell; the expressed NMDAR protein or protein subunit thereof is present on the cell surface; such that the mammalian cell is viable.
  • the cell viability is measured by methods known in the art to show viability, such as - in a thirty-second embodiment - by measuring the amount of adenosine triphosphate (ATP) present, which indicates the presence of metabolically active cells.
  • ATP adenosine triphosphate
  • the transiently expressing NMDAR protein or protein subunit thereof on the surface of the mammalian cell is by a transient transfection of the mammalian cell with a vector and expression of an NMDAR gene on the vector.
  • the vector is a baculovirus vector containing an NMDAR gene, such as by the infection or transduction of a BacMam vector.
  • the NMDAR is a human NMDAR. In a thirty-sixth embodiment, the NMDAR is a mouse NMDAR.
  • the invention further provides a method for producing a viable mammalian cell having NMDAR on its surface.
  • the method includes the steps of: (a) transiently transfecting the mammalian cell with a vector selected from (i) a baculovirus vector containing one or more expressible NMDAR genes, or a plasmid coding for NMDAR; and (b) culturing the mammalian cell, where the culture medium (i) contains an NMDAR ligand binding site antagonist; and (ii) does not contain an NMDAR channel blocker.
  • transiently expressing NMDAR protein or subunit on the surface of the mammalian cell is accomplished by a transient transfection of the mammalian cell with a vector and expression of an NMDAR gene on the vector.
  • the vector is a baculovirus vector containing an NMDAR gene, such as a BacMam vector.
  • the mammalian cell in the assay is a human cell, such as, in a forty-first embodiment. HEK293, or in a forty-second embodiment, a human iPSC.
  • the invention provides a method for facilitating the sensitivity of NMDAR to ligands of the NMDAR glycine site or to the NMDAR glutamate site.
  • the method includes the steps of (a) transiently expressing NMDAR protein or protein subunits thereof on the surface of a mammalian cell; (b) culturing the mammalian cell in a culture medium, where the culture medium contains an NMDAR ligand binding site antagonist; but does not contain an NMDAR channel blocker; (c) then, while retaining the mammalian cell, replacing (i) the culture medium that contains the
  • NMDAR ligand binding antagonist and does not contain an NMDAR channel blocker with (ii) a buffer or culture medium that does not contain an NMDAR channel blocker; (d) contacting the mammalian cell with a test agent; (e) contacting the mammalian cell with an NMDAR ligand site binder; and (f) determining whether the test agent binds to the glycine binding site or the glutamate binding site.
  • transiently expressing NMDAR protein or subunit on the surface of the mammalian cell is accomplished by a transient transfection of the mammalian cell with a vector and expression of an NMDAR gene on the vector.
  • the vector can be a baculovirus vector containing an NMDAR gene, such as a BacMam vector.
  • the method further includes, after the culturing step, manipulating the mammalian cell to respond in a measurable way to changes in calcium concentration in the mammalian cell.
  • the method further includes, both after the contacting step (d) and after the contacting step (f), measuring the calcium response by the mammalian cell.
  • the invention provides a method for dissecting the mechanism of action of NMDAR inhibitors.
  • the method includes the steps of (a) transiently expressing NMDAR protein or protein subunits on the surface of a mammalian cell; (b) culturing the mammalian cell in a culture medium that contains an NMDAR ligand binding antagonist, but does not contain an NMDAR channel blocker; then (c) manipulating the mammalian cell to respond in a measurable way to changes in calcium concentration in the mammalian cell; (d) while retaining the mammalian cell, replacing the culture medium that contains the NMDAR ligand binding antagonist and does not contain an NMDAR channel blocker with a buffer or culture medium that does not contain an NMDAR channel blocker; (e) measuring the calcium response by the mammalian cell; (f) contacting the mammalian cell with a test agent; (g) measuring the calcium response by the mammalian cell to the test agent; (
  • transiently expressing NMDAR protein or protein subunit on the surface of the mammalian cell is accomplished by a transient transfection of the mammalian cell with a vector and expression of an NMDAR gene on the vector.
  • the vector can be a baculovirus vector containing an NMDAR gene, such as a BacMam vector containing an NMDAR gene.
  • the invention provides a method for detecting calcium levels in a eukaryotic cell.
  • the method includes the steps of: (a) transfecting the eukaryotic cell with a polynucleotide that contains an expressible gene encoding a calcium sensor (GECS)-based GCaMP-protein; (b) expressing the gene for a genetically encoded calcium sensor to create a eukaryotic cell stably expressing GCaMP protein; (c) contacting the eukaryotic cell with a test agent; and (d) measuring the calcium response by the eukaryotic cell to the test agent.
  • GECS calcium sensor
  • the polynucleotide is a lentivirus.
  • the expressible gene encodes GCAMP6, with two aspartic acid (D) -> cysteine (C) point mutations (dCys-GCAMP6; SEQ ID NO: 1).
  • the method of detecting calcium levels in mammalian cells uses a genetically encoded calcium sensor (GECS) based GCaMP- protein, encoded by an expressible gene in a recombinant lentivirus and stably expressed in cells.
  • the expressible gene can be one of the GCaMP6 calcium sensors with two aspartic acid (D) -> cysteine (C) point mutations (dCys-GCAMP6; SEQ ID NO: 1), which showed increased signal and sensitivity to calcium in biochemical assays and in neuronal imaging, by decreasing the background and unspecific signal due to dead cells and exposure to extracellular calcium.
  • D aspartic acid
  • C cysteine point mutations
  • GCaMP6s have not generally been used to generate a plate-based readout of calcium levels in cells.
  • GCaMP6s with the aspartic acid (D) -> cysteine (C) point mutations (dCys- GCAMP6; SEQ ID NO: 1) have also not generally been used.
  • the calcium level detection method of the invention combines all three elements (stable expression allowing for done selection, use of the improved GCaMP6s and introduction of GCaMP mutations).
  • the calcium level detection method of the invention provides for the detection of calcium levels in cells without the need to use a calcium sensitive dye, thus providing cost savings and time savings and without the need to incubate the cells with the toxic dye anymore which might lead to false results due to cell stress, and making available a high throughput screen.
  • the calcium level detection method of the invention can be used for cell imaging or in many assay formats, including the NMDAR assays of the invention.
  • the invention provides assay and methods useful in screening for modulators of NMDARs, such as positive allosteric modulators ("PAMs") of NMDARs for use in in treating or palliating schizophrenia or other NMDAR-based indications.
  • This invention provides an assay to identify low molecular weight, brain-penetrant, NR2A-selective, positive allosteric modulators (PAMs) of the NMDAR.
  • PAMs positive allosteric modulators
  • An advantage of PAMs is that they would enhance NMDAR activity exclusively when the receptor is activated by endogenously-released co-agonists (glutamate and D-serine or glycine), in response to physiological synaptic activity.
  • An NR2A-specific PAM would allow engagement of a subpopulation of NMDARs thereby increasing the safety margin. Normalization of NMDAR activity in the brain of patients suffering from schizophrenia is predicted to mitigate positive, negative and cognitive symptoms thereby addressing unmet medical needs in schizophrenia.
  • the method includes the steps of (a) transiently expressing NMDAR proteins NR1 and NR2, or the subunits, on the surface of a mammalian cell; (b) culturing the mammalian cell in a culture medium that contains an NMDAR ligand binding antagonist; and does not contain an NMDAR channel blocker; (c) manipulating the mammalian cell to respond in a measurable way to changes in calcium concentration in the mammalian cell; (d) measuring the calcium response by the mammalian cell; (f) contacting the mammalian cell with a test agent; (g) measuring the calcium response by the mammalian cell to the test agent; (i) contacting the mammalian cell with an NMDAR ligand site binder; and (j) again measuring the calcium response by the mammalian cell.
  • An increase in calcium response by the mammalian cell identifies the test agent as a positive allosteric modulator of NMDARs for use in treating
  • the test agent to palliate schizophrenia is further assayed by (b) measuring tyrosine phosphorylation of the NR2 subunit of the NMDAR of the cell in the presence and absence of the test agent, where an increase in tyrosine phosphorylation of the NMDAR NR2 subunit in the presence of the test agent identifies an agent as a candidate compound to palliate schizophrenia.
  • assaying a test agent for electrophysiological effect upon interaction with a human CNS receptor can be done by determining ligand-induced electrical current across said cell or membrane. See, U.S. Pat. No. 6,500,634 (Foldes et ai.).
  • transiently expressing NMDAR protein or protein subunit on the surface of the mammalian cell is accomplished by a transient transfection of the mammalian cell with a vector and expression of an NMDAR gene on the vector.
  • the vector can be a baculovirus vector containing an NMDAR gene, such as a BacMam vector containing an NMDAR gene.
  • One advantage of the assay and methods of the invention is in discontinuing the use of ketamine in in vitro assays, which is an art-recognized problem to be solved. Cik et ai., Biochem. J. 296:877-83 (1993). To prevent NMDAR-mediated cytotoxicity, previous studies have inhibited NMDAR activity by culturing cells in culture media containing ketamine or similar NMDAR channel blockers. These blockers are difficult to remove from the cells, even by extensive washing before the assay. Moreover, ketamine is a restricted substance, due to its abuse as a recreational drug.
  • Another advantage of the assay and methods of the invention is speed.
  • the assay of the invention is efficient and can be used to measure NMDAR activity in a glycine-sensitive and glutamate-sensitive manner in a plate-based fluorescent assay in less than 24 hours.
  • the method of the invention requires only a short incubation time, a total of about 16 hours in sixty-second embodiment.
  • the setup of the assay of the invention does not require a clone selection step.
  • Another advantage of the assay and methods of the invention is that the expression and stoichiometry of NMDAR subunits is adjustable and titratable.
  • Another advantage of the assay and methods of the invention is sensitivity to both glycine and L-glutamate.
  • the assay and methods of the invention make available to neuroscientists and biochemists the rapid and flexible assessment of both glycine and glutamate sensitivity of NMDAR in the same cells.
  • Previous cell models have been sensitive to glutamate but not to glycine.
  • Glycine is present at saturating amounts in culture media, and is produced and secreted by the mammalian cells themselves.
  • the method of the invention includes a step of culturing cells in the presence of a weak glycine binding-site antagonist (MDL105,519), which can be easily washed off the cells in a later step of the method.
  • MDL105,519 weak glycine binding-site antagonist
  • the system of the invention shows endogenously high levels of glutamate. L-glutamate sensitivity can be assayed by the addition of a weak glutamate binding site antagonist.
  • Another advantage is the increased sensitivity of the assay due to a lowering or the background fluorescence caused by the calcium 6 dyes.
  • Another advantage is the removal of the dye loading step, which is toxic for some cell types and further increases the background signal and hence decreases sensitivity. Another advantage is that there is no need to add probenecid, which can also interfere with assay of the invention or similar assays.
  • Another advantage of the assay and methods of the invention is that it makes available to neuroscientists and biochemists the measurement of D-serine effect on
  • NMDARs D-serine -binds to the same NMDAR binding site as glycine. L-serine is much more abundant than D-serine, and the high L-serine levels have previously made it difficult to measure D-serine levels with classical biochemical methods.
  • the system of the invention now makes available to neuroscientists and biochemists readout for D- serine levels generated in biochemical reactions or cell supernatants, even in the presence of L-serine.
  • NMDARs are ⁇ 200-fold less sensitive to L-serine than D-serine , which now makes available the analysis of D-serine levels in the presence of L-serine.
  • An unexpected result of the assay and methods of the invention is that it is necessary to block ligand binding of native NMDAR during the culturing step.
  • data generated in the practice of the invention show that it is not sufficient to only block NMDAR-mediated toxicity in the cells (e.g., with ketamine, as in previous assays). It is also necessary to block ligand binding to the native receptors during the mammalian cell culture. The practice of the invention does not result in a complete rescue of cell viability, but does result in a much better signal in the activity assay.
  • the assay of the invention can be used to identify inhibitors of NMDAR (competitive, noncompetitive, and allosteric).
  • the method can be used to assess the effect of the test agent at different activation levels of NMDAR (positive allosteric modulator assays are run at sub-maximal ligand concentrations, whereas inhibitor assays are run at EC80-100 ligand concentration).
  • the assay can be used to identify compounds that show ligand-specificity, i.e. they only work in the presence of on ligand but not the other.
  • FIG. 1 is an immunoblot showing the expression of NMDAR in mammalian cells using baculovirus.
  • HEK293 cells in the presence of 1 mM ketamine were transduced with different amounts of baculovirus vector encoding human NR1 and NR2A. The cells were harvested 16 hours after transduction.
  • NR1 and NR2A protein levels were analyzed by immunoblotting.
  • FIG. 1 shows NR1 and NR2A protein levels in HEK293 cells transduced with baculovirus. Representative images are shown. This figure supports the finding that the method of the invention is titratable.
  • FIG. 2 is a model showing the prior art lack of NMDAR activity in functional assay after protection with MK801 or ketamine.
  • NMDAR-ligands contained or secreted by the cells into the media activate NMDAR and lead to excitotoxicity. Protection with MK801 or ketamine prevent excitotoxicity, but are difficult to wash out and do not prevent occupation of the ligand binding sites with endogenous ligand, both of which prevent NMDAR activity in functional assays.
  • FIG. 3 is a model of the protection facilitated sensitivity of NMDAR activity to glycine ("Protection with glycine binding antagonist MDL105.519 (Glycine-sensitive mode)") and glutamate ("Protection with glutamate binding antagonist CGP070667 (Glutamate-sensitive mode)").
  • the addition of ligand binding site antagonists protects NMDAR by occupying the ligand binding site and preventing the binding of endogenous ligands. After washout of the ligand binding site antagonist, the ligand binding sites become accessible for exogenously added ligand, thus facilitating ligand-sensitivity.
  • FIG. 4 is a plasmid map of the Gateway compatible BacMam vector prior to recombination to produce final expression vectors.
  • 7-CKA is 7-chloro-4-oxo-1 H-quinoline-2- carboxylic acid, is a weak competitive NMDAR glycine binding site antagonist, CAS Number 18000-24-3.
  • 7-CKA is commercially available from Sigma-Aldrich, St. Louis MO USA, and from Tocris Bioscience, Minneapolis MN USA.
  • AAM077 is [[[(1 S)-1 -(4-Bromophenyl)ethyl]amino](1 ,2,3,4-tetrahydro-2,3-dioxo- 5-quinoxalinyl)methyl]phosphonic acid tetrasodium hydrate, CAS Registry Number 459836-30-7, a NR2A-selective NMDAR antagonist. See, Frizelle P et al., Mol.
  • AAM077 is commercially available from Calbiochem, Billerica MA USA.
  • AP5 (2-amino-5-phosphonopentanoic acid, APV) is a competitive glutamate binding site antagonist.
  • the CAS Registry Number is 76326-31 -3 AP5 is commercially available from Sigma-Aldrich, St. Louis MO USA, and from Tocris Bioscience,
  • AP5 was not sufficient as an NMDAR ligand binding site antagonists to protect the cells from excitotoxicity.
  • BocMam a Baculovirus gene transfer into Mammalian cells system, is a use of modified insect cell virus (baculovirus) as a vector to deliver and express genes in mammalian cells. See, U.S. Pat. No. 5,871 ,986 (Boyce), Hofmann & Strauss,
  • BacMam can be purchased from Thermo Fisher Scientific, Waltham MA USA.
  • a "bacmid” is a baculovirus shuttle vector that can be propagated in both E. coli and insect cells.
  • a "baculovirus vector” is a covalently closed circular double stranded
  • Baculovirus vectors are based upon Baculoviridae viruses, which infect arthropods as their natural hosts.
  • the baculovirus vectors mainly used as expression vectors are based upon Autographa californica nucleopolyhedrovirus (NPV), which was isolated from alfalfa looper larva.
  • NPV Autographa californica nucleopolyhedrovirus
  • Examples of commercially available baculovirus vectors include the BacMam system (see above), the Baculovirus Expression Vector System (from BD Biosciences, San Diego CA USA) and Baculovirus Expression System - BacPAK (from Clontech Laboratories, Inc., Mountain View CA USA).
  • a recombinant baculovirus vector can contain one or more nucleotide sequence of a human NMDAR NR1 subunit and/or NR2 subunit together with additional sequences capable of directing the synthesis of the human NMDAR subunit in cultures of transiently transfected mammalian cells. See, EP0672140B1 (Merck Sharp & Dohme, Ltd.).
  • a "calcium sensor protein” is a protein that can be expressed inside living (viable) cells and responds in a measurable way to changes in calcium concentration.
  • Examples of calcium sensor proteins include the GCaMPs.
  • Calcium 6 dye is a proprietary fluorophore that provides a high quantum yield compared to other calcium indicators, for use with a FLIPR® calcium assay kit.
  • FLIPR® Assay Kits are commercially available from Molecular Devices, LLC, Sunnyvale CA USA. Similar calcium dyes for use in FLIPR® Assay Kits from Molecular Devices include calcium 5 dye, calcium 4 dye and calcium 3 dye.
  • a "cell-permeable calcium indicator” is a chemical compound that can pass from culture media or solutions through the membranes of cells, where they respond in a measurable way to changes in calcium concentrations in living or viable cells.
  • Examples of cell-permeable calcium indicators are fura-2, lndo-1 , calcium 3 dye, calcium 4 dye, calcium 5 dye and calcium 6 dye.
  • Methods of measuring changes in calcium concentration in the mammalian cell are known in the neurobiological arts. For example, primary cultures of rat cerebellar granule cells loaded with the fluorimetric indicator fura-2 were used to measure changes in [Ca 2+ ] elicited by NMDA and its co-agonist glycine. See, WO 95/21612 (NPS Pharma, Inc.).
  • CellTiter-Glo® a Luminescent Cell Viability Assay, is a method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.
  • the CellTiter-Glo® Assay is available from
  • CGP039653 is an NMDAR ligand. See, Auberson YP et a/. , Bioorg. Med. Chem. Lett. 12: 1099-1 102 (2002).
  • CGP070667 is ((2,3-dioxo-1 ,2,3,4-tetrahydroquinoxalin-6-yl)methyl)phosphonic acid. See, Auberson YP et a/. , Bioorg. Med. Chem. Lett. 12: 1099-1 102 (2002).
  • CIQ is (3-chlorophenyl)(6,7-dimethoxy-1 -[(4-methoxyphenoxy)methyl]-3,4- dihydroisoquinolin-2(1 H)-yl)methanone, CAS Registry Number 486427-17-2. See, Zhang X et al., British J. Pharmacol. 171 (16): 3938-3945 (March 2014). CIQ is commercially available from Tocris Bioscience, Minneapolis MN USA.
  • CPP is 3-(2-carboxypiperazin-4-yl)propyl-1 -phosphonic acid, a selective NMDAR antagonist.
  • CPP can be purchased as a ( ⁇ )-CPP solid from Sigma-Aldrich Corp., St. Louis MO USA.
  • CP101606 is (1 S, 2S)-1 -(4-hydroxyphenyl)-2-(4- hydroxy-4-phenylpiperidino)-1 -propanol, a noncompetitive antagonist of NMDARs containing the NR2B subunit. See, Menniti FS et al., Neuropharmacol. 39(7):1 147-55 (27 April 2000). CP-101 ,606 is produced by Pfizer (Groton CN USA).
  • dCys-GCAMP6s (SEQ ID NO: 1) is a dye showing similar sensitivity as calcium6 dye.
  • the mutations introduced into dCys-GCAMP6s increase sensitivity as compared to Calcium6 dye.
  • the specific mutations render dCys-GCAMP6s insensitive to calcium under oxidative conditions (extracellular environment). This results in a reduction of unspecific signal due to loss of membrane integrity (e.g., dead cells).
  • Cells containing dCys-GCAMP6s show similar to higher sensitivity and higher signal range than cells loaded with Calcium6 dye.
  • the advantages of using dCys-GCAMP6s include (a) cost; (b) time; (c) sensitivity; (d) flexibility; and (e) opportunities for multiplexing
  • FDSS is a Functional Drug Screening System.
  • FDSS ⁇ CELL is a kinetic plate reader with an integrated dispensing head and imaging-based detector, allowing for
  • FDSS ⁇ CELL can be purchased from Hamamatsu Photonics K.K., Hamamatsu City, Japan. Hamamatsu also provides a 1536 well format. The system allows for online liquid addition and is compatible with classical fluorescent dyes for calcium imaging.
  • GCaMP is a genetically encoded calcium indicator (GECI), created from a fusion of green fluorescent protein (GFP), calmodulin, and M13, a peptide sequence from myosin light chain kinase. Nakai et al., Nature Biotechnol. 19:137-141 (2001).
  • GCaMPs have been modified to improve the range of the fluorescence signal, resulting in GCaMP3 (Tian et. al., Nat. Methods 6:875-881 (2009)) through GCaMP8 (Ohkura et al., PLoS ONE 7(12): 051286 (2012)). Red fluorescence GECIs have also been developed. Zhao et ai, Science 333:1888-1891 (201 1).
  • GCaMP6 is a calcium sensor (GECS) based GCaMP protein. See, Chen et al., Nature 499(7458): 295-300 (18 July 2013). GCaMP6 and vectors containing the
  • GCaMP6 gene are commercially available from Addgene (Cambridge MA USA).
  • dCys-GCAMP6s shows similar sensitivity as calcium 6 dye and higher signal range.
  • the mutations increase sensitivity to calcium by rendering the protein probe insensitive to calcium under oxidative conditions (such as extracellular environment), which leads to reduction of unspecific signal due to loss of membrane integrity (as in dead cells).
  • Glutamate is a naturally-occurring amino acid. NMDARs have a glutamate binding site.
  • Glycine or "Gly” is a naturally-occurring amino acid. NMDARs have a glycine binding site.
  • GNE-6901 is 7-(4-fluorophenoxymethyl-3-[trans-2-hydroxymethyl)cycloproyl]-2- methyl-5H-[1 ,3]thiazolo[3,2-a]pyrimidin-5-one (enantiomer 2), as described in
  • HBSS Hank's Balanced Salt Solution, a balanced salt solution rich in bicarbonate ions, made to a physiological pH (roughly 7.0-7.4) and salt concentration, maintain mammalian cells' pH and osmotic balance. Originally prepared by Hanks & Wallace, Proc. Soc. Exp. Biol. Med. 71 :196-200 (1949), HBSS is now available in several formulations and formats. HBSS is often used for washing cells, as a diluent, or as an inorganic base for standard media preparations. HBSS can be purchased from commercial sources, including from Thermo Fisher Scientific, Waltham MA USA.
  • ⁇ cells are Human Embryonic Kidney 293 cells, which are derived from cells originally generated in 1973 by transformation of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA.
  • HEK cells are available from commercial sources, including from the American Type Culture Collection (ATCC), Manassas VA USA.
  • a "high-throughput screen” (“UTS”) is a drug-discovery process widely used in the pharmaceutical industry that uses automation, robotics, data processing and control software, liquid handling devices, and sensitive detectors to quickly assay the biological or biochemical activity of a large number of drug-like compounds. See, “High-Throughput Screening Challenges”. Genetic Engineering & Biotechnology News. Drug Discovery Roundtable Discussion 28(14): 26-27. (Mary Ann Liebert, 01 August, 2008).
  • Ifenprodil is 4-[2-(4-benzylpiperidin-1 -yl)-1 -hydroxypropyl]phenol, a selective inhibitor of NMDARs composed of the NR1/2B subunits. See, Reynolds & Miller, Mol. Pharmacol. 36(5): 758-65 (1989).
  • Ketamine is the NMDAR channel blocker/non-competitive inhibitor (RS)-2-(2- chlorophenyl)-2-(methylamino)cyclohexanone). Ketamine is a dissociative psychedelic with antidepressant properties used as an anesthesia in humans and animals, and is also used recreationally for its effects on the central nervous system, and thus regulated as a Controlled Substance in many countries. Ketamine's use as a recreational drug has been implicated in several deaths worldwide.
  • RS non-competitive inhibitor
  • L701 ,324" is 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1 H)-quinolinone, a selective NMDAR glycine site antagonist. Obrenovitch & Zilkha, British J. Pharmacol. 1 17: 931 -937 (1996).
  • Lentivirus vectors or "lentiviral vectors” are a type of retrovirus that are adapted as gene delivery vehicles (vectors) in the biochemical arts and that can infect both dividing and non-dividing cells.
  • vectors gene delivery vehicles
  • MDL105.519 (CAS Registry Number 161230-88-2) is a competitive NMDAR glycine binding site antagonist and an inhibitor of NMDAR-mediated responses in vitro and in vivo. MDL105.519 is commercially available from PerkinElmer, Waltham MA USA.
  • Memantine is the NMDAR channel blocker 3,5- dimethyltricyclo[3.3.1 .13,7]decan-1 amine (CAS Registry Number 19982-08-2).
  • Memantine is commercially available from several pharmaceutical companies, including Forest Laboratories, New York NY USA, as Namenda®.
  • MK801 or "dizocilpine” is the NMDAR channel blocker, [5R,10S]-[+]-5-methyl- 10,1 1 -dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, CAS Registry Number 77086-21 - 6. See, U.S. Pat. Nos. 4,399,141 (Anderson et ai.) and 4,477,668 (Bender et a/.). MK801 has been used as a recreational drug. MK801 is available from commercial sources, including from Calbiochem, Billerica MA USA.
  • a "modulator of an N-methyl-D-aspartate receptor (NMDAR) function” is a compound that modulates the activity of an NMDAR protein or NMDAR subunit.
  • a modulator of NMDAR function can be an uncompetitive channel blocker; a noncompetitive antagonist, partial agonist or agonist; or a competitive antagonist, partial agonist or agonist. See, examples in Van Dongen AM, editor. Biology of the NMDA Receptor. (CRC Press/Taylor & Francis, Boca Raton FL USA, 2009).
  • a modulator of NMDAR function can be determined by the method or assay of the invention.
  • NMDAR N-methyl-D-aspartate receptor
  • NMDA receptor NMDAR
  • NMDAR protein is a glutamate receptor and ion channel protein found, e.g., in nerve cells.
  • NMDAR includes seven different receptor subunits.
  • GRIN1 subunit which can combine with GRIN2 subunits (GRIN2A, GRIN2B, GRIN2C, and GRIN2D) and/or GRIN3 subunits (GRIN3A and GRIN3B).
  • Combinations of these subunits can form either di- heteromeric (e.g., 2 units of GRIN1 and 2 GRIN2A subunits) or more complex tri- heteromeric (e.g., 2 units of GRIN1 and 1 GRIN2A, 1 GRIN2B) receptor complexes.
  • NMDARs mediate the transfer of electrical signals between neurons in the brain and in the spinal column. For electrical signals to pass, the NMDAR must be open. To remain open, glutamate and glycine must bind to the NMDA receptor. An NMDAR that has glycine and glutamate bound to it and has an open ion channel is "activated.”
  • NMDAR antagonists are a class of anesthetics that work to antagonize, deactivate or inhibit the action of NMDAR. Chemicals that block the function of the NMDAR are called antagonists. NMDAR antagonists fall into four categories: competitive antagonists (which bind to and block the binding site of the neurotransmitter glutamate); glycine antagonists (which bind to and block the glycine site); noncompetitive antagonists (which inhibit NMDARs by binding to allosteric sites); and uncompetitive antagonists (which block the ion channel by binding to a site within it). See, Kim AH et ai, "Blocking Excito toxicity". In Marcoux FW & Choi DW. CNS Neuroprotection, pp. 3-36 (New York: Springer, 2002).
  • NMDAR channel blockers are uncompetitive or non-competitive NMDAR antagonists that block the open ion channel, particularly after activation by agonists.
  • Examples of NMDAR channel blockers include ketamine and dizocilpine (MK801).
  • MK801 ketamine and dizocilpine
  • Some NMDAR channel blockers, such as ketamine are recreational drugs used for their dissociative, hallucinogenic, and euphoriant properties.
  • NMDAR glutamate binding site antagonists are compounds that bind to glutamate binding site on NMDARs. Glutamate ligand binding site antagonists include the protective compound CGP070667.
  • NMDAR glycine binding site antagonists are compounds that bind to glycine binding site on NMDARs.
  • Glycine binding site antagonists include the protective compound MDL105,519.
  • NMDAR ligand binding site antagonist is a compound that binds to the glutamate binding site or glycine/D-serine binding site of NMDAR to antagonize NMDAR activity.
  • the NMDAR ligand binding site antagonist can be MDL105,519 or CGP070667.
  • NMDAR ligand site binder is a compound that binds to the glutamate or glycine/D-serine binding site of NMDAR to affect NMDAR, particularly in the later stages of the method of the invention.
  • NMDAR ligand site binder can bind to NMDAR glutamate sites or NMDAR glycine sites, and can be positive or negative effectors of NMDAR activity.
  • the NMDAR ligand site binder can be CGP070667, AAM077, 7-CTKA (7-CKA), MDL105,519, L701 .324, CGP039653 or CPP.
  • NR1/2A "NR1 /NR2A” or “GNIR1 /NR2A” is an NMDAR comprising only subunits NR1 (glycine binding NMDA receptor subunit 1 ; GluN1) and NR2A (glutamate binding NMDA receptor subunit 2; GluN2A) subunits.
  • NR1/2B is an NMDAR comprising only subunits NR1 (glycine binding NMDA receptor subunit 1 ; GluN1) and NR2B (glutamate binding NMDA receptor subunit 2; GluN2B) subunits.
  • NR1 /2B "NR1 /NR2D” or “GNIR1 /NR2D” is an NMDAR comprising only subunits NR1 (glycine binding NMDA receptor subunit 1 ; GluN1) and NR2D (glutamate binding NMDA receptor subunit 2; GluN2D) subunits.
  • Plasmids are small DNA molecules within a cell that is physically separated from a chromosomal DNA and can replicate independently. Plasmids are most commonly found in bacteria as small, circular, double-stranded DNA molecules;
  • EXAMPLE 3 for the transient expression of NMDAR in HEK293 cells, approximately 2 ⁇ g DNA (as plasmids pcDNA 1 -Amp/hNR2A or pcDNA 1 -Amp/hNR 1 - 3C) was transfected per 10 5 HEK293 cells, by lipofectin-mediated DNA transfection according to the manufacturer's (Life Technologies Inc., Gaithersburg MD USA) specifications. In co-expression experiments, i.e.
  • the HEK293 cells were similarly transfected with 3 ⁇ g of a DNA mixture containing pcDNAI -Amp/hNR2A and pcDNA1 -Amp/hNR1 -3C. Briefly, HEK293 cells were plated at a density of 10 5 cells/dish and then grown for 24 hours in 10% FBS- supplemented MEM medium (Life Technologies Inc., Gaithersburg MD USA). The medium was then removed and cells were washed in OPTI-MEM I medium (Life Technologies Inc., Gaithersburg MD USA) lacking FBS, prior to transfection.
  • a transfection solution (100 ⁇ ) containing 5-7.5 ⁇ of lipofectin and DNA was then applied to the cells. After incubation for six hours at 37°C, cells were washed and then allowed to grow for 36-48 hours in 10% FBS-supplemented MEM medium containing 50 ⁇ glutamate binding site antagonist AP5 prior to electrophysiological recording.
  • Other slightly different protocols, using different reagents like FuGene6, are known to those of skill in the art.
  • Probalan is 4-(dipropylsulfamoyl)benzoic acid, CAS Registry Number 57-66- 9, sold under the brand name Probalan.
  • QNZ46 is 4-[6-methoxy-2-[(1 E)-2-(3-nitrophenyl)ethenyl]-4-oxo- 3(4H)quinazolinyl]benzoic acid. See, Mosley et al., J. Med. Chem. 53 5476 (2010); Hansen & Traynelis, J. Neurosci. 31 : 3650 (201 1 ). QNZ46 is commercially available from Tocris Bioscience, Minneapolis MN USA.
  • R025-6981 is (af?,pS)-a-(4-Hydroxyphenyl)-p-methyl-4-(phenylmethyl)-1 - piperidinepropanol, a potent and selective activity-dependent blocker of NMDA receptors containing the NR2B subunit. See, Fischer et al., J. Pharmacol. Exp. Ther. 283: 1285 (1997). R025-6981 is commercially available from Tocris Bioscience, Minneapolis MN USA.
  • TN201 is 3-chloro-4-fluoro-N-[4-[[2-
  • TCN201 is an allosteric negative modulator of NMDARs.
  • TCN201 is commercially available from Tocris Bioscience, Minneapolis MN USA.
  • TCN201 selectively blocks N2A-containing NMDARs in a N1 co- agonist dependent, but non-competitive manner. Edman S et al., Neuropharmacology (2012). In the assay of the invention (TABLE 4), TCN201 was not found to be sufficient as an NMDAR ligand binding site antagonist to protect the cells from excitotoxicity.
  • test agent is an agent that is suspected of being a modulator of NMDAR function and thus a candidate for testing by the method of the invention.
  • a test agent can be suspected of being a modulator of NMDAR function based upon its activity in a different assay than the assay of the invention, based upon its chemical structure, or for any other reason.
  • a "viable" cell is a ceil, whether in vivo or in vivo, that is alive. Viability of a cell can be determined by any means known to those in the neurobiological or biochemical arts, such as by quantitation of the ATP present in cells, which is an indicator of metabolically active cells.
  • One assay for quantitation of the ATP present in cells is a CellTiter-Glo® assay. See, TABLE 2.
  • NMDARs have been studied by neuroscientists and biochemists because of their involvement in synaptic plasticity and cognition. Genetics and functional studies have implicated glutamate and NMDARs in schizophrenia. Gordon JA, "Testing the glutamate hypothesis of schizophrenia", Nat. Neurosci. 13, 2-4 (2010). NMDARs have been implicated in other nervous system disorders such as epilepsy, stroke, pain, addiction, depression and Alzheimer's disease. NMDARs may also be targets for diseases in other organ systems such as diabetes (Marquard et al, Nature Medicine, 21 (4): 363 (2015)), in regulation immune function (Simma et al., Cell Commun. Signal. ,12: 75 (2014); Farooq et al., Am. J. Physiol. Gastrointest. Liver Physiol., 307(7): G732-40 (2014)),
  • NMDAR modulators The inventors wanted to identify and develop NMDAR modulators, to treat or palliate schizophrenia and other NMDAR-related diseases. We recognized the challenge developing such NMDAR modulators, given the experience of others in the
  • HTS high- throughput screening
  • PV-INs parvalbumin-positive class of inhibitory interneurons
  • Post-mortem schizophrenia brain analyses also show a decrease in dendritic spine density in glutamatergic neurons, enhanced neuregulin-erb4 signaling (known to suppress NMDAR function), reduced levels of NR2A-containing NMDARs and a decreased density of NR2A-positive GABAergic interneurons.
  • These studies highlight a critical role for glutamate signaling, likely via NR2A-containing NMDARs in SZ pathophysiology.
  • Enhancement of NMDAR activity with agents that target the glycine modulatory site directly e.g. glycine, D-serine, alanine
  • indirectly sarcosine
  • the invention provides a different approach to studying the pharmacology of NMDARs.
  • the unbiased identification of weak glycine and glutamate site antagonists that protect cells from NMADR toxicity and the ability to readily remove these compounds were important in the development of the assay of the invention.
  • These compounds compete out the exogenous and saturating levels of ligand (L-glutamate or glycine) that maintain the receptor in an activated, presumably desensitized state. Once these compounds are removed, the glycine/D-serine site is no longer saturated, enabling the study of different co-agonist concentrations on calcium signaling with much less background interference.
  • the assay of the invention is biologically relevant. We were able to recapitulate the EC 50 s for the agonist glutamate and the co-agonists glycine or D-serine . We were able to faithfully recapitulate the effects of several known NMDARs inhibitors and allosteric modulators in our assay. See TABLE 17.
  • the assay of the invention also shows the expected sensitivity to magnesium. With depolarized membrane potentials, the assay of the invention becomes less sensitive to magnesium. See, TABLE 1 1 .
  • the method of the invention uses BacMam to introduce different receptor subunits.
  • BacMam vector in combination with the use of weak glycine and glutamate site antagonists, allows neuroscientists and biochemists to study distinct pharmacological activities of small molecules such as the identification of co-agonist glycine or D-serine molecules vs. glutamate as well as to test the effect of small molecules in different receptor subunits (di-heteromeric or tri-heteromeric).
  • Baculovirus vectors provide a gentle, titratable way of transfecting mammalian cell lines and primary cells. For assays in which expression from multiple different subunits are compared, expression from individual baculovirus vectors offers the advantage of individually titratable expression and the ease of switching protein subunits using individually encoding viruses. See, FIG. 1 .
  • Alternatives to the BacMam system include the traditional lipofectamine or Fugene-based transient transfection systems.
  • Expression of the NMDAR may also be accomplished by a variety of different promoter-expression systems in a variety of different host cells. See, e.g., EP 0 672 140 B1 (Merck Sharp and Dohme, Ltd.), which discloses transiently transfected cells containing NMDAR.
  • the eukaryotic host cells suitably include yeast, insect and mammalian cells. Suitable mammalian host cells include rodent fibroblast lines, e.g. , mouse Ltk " , Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; and HEK293 cells.
  • the assay and methods of the invention provide for rapid comparisons of different NMDAR subunits.
  • the assay and methods of the invention now make available to neuroscientists and biochemists a way to measure the effects of agonists for both the glycine binding site (e.g., glycine or D-serine ) and the glutamate binding site (e.g., glutamate or NMDA) in different combinations.
  • the assay and methods of the invention do not use channel blockers such as ketamine or MK-801 to mitigate against cellular toxicity and maintaining the ligand binding site in a ligand-free state. Instead, the assay and methods of the invention leverage the use of weak NMDAR glycine binding site antagonists or weak NMDAR glutamate binding antagonists, which now can be identified in an unbiased screen.
  • weak NMDAR glycine binding site antagonists or weak NMDAR glutamate binding antagonists are readily washed off, resulting in the ability to measure reliable calcium traces following activation with different concentrations of ligand and/or ligand combinations.
  • the use of weak antagonists now makes available to neuroscientists and biochemists a way to not only maintain cells alive during the assay, but also to study the activation of NMDARs by different ligands, with sensitivity to both D-serine /glycine and glutamate/NMDA.
  • the assay and methods of the invention provide neuroscientists and biochemists with the ability to investigate the effects of small molecules on the activation of NMDARs using different ligands and ligand binding sites.
  • the assay system of the invention replicates the pharmacology of NMDAR in response to known antagonists, allosteric modulators, as well as sensitivity to magnesium.
  • NMDAR expression in cells in vitro is cytotoxic
  • we the inventors began efforts to develop an NMDAR assay that does not have the limitations of currently available models, which have (a) high expression levels of NMDAR NR2 subunit; (b) a random ratio of NMDAR subunits; (c) a need to use ketamine or similar strong NMDAR channel blockers for protection excitotoxicity, and (d) a lack of sensitivity to glycine/D- serine (and a limited sensitivity to glutamate) in a plate based assay, due to the high levels of glycine in cell media, including glycine and other amino acids secreted by dying cells in culture.
  • This method provides a transient, fast, flexible assay, with low expression levels to minimize cytotoxicity.
  • the assay is an unbiased screen for optimal protection compounds.
  • the pACT vector has a strong cytomegalovirus ("CMV") promoter region which includes the CMV immediate early promoter, plus a chimeric intron comprised of the 5 ' -donor site from the first intron of the human ⁇ -globin gene and the branch and 3 ' -acceptor site from the intron of an immunoglobulin gene heavy chain variable region.
  • CMV cytomegalovirus
  • the resultant BacMam vector contained the strong CMV promoter and a mammalian-two-hybrid fusion protein.
  • This strong promoter BacMam vector was Gateway-adapted by cloning a Gateway® cassette into an Xhol site of the vector. See, the Xhol site in FIG. 4. This removed the pACT-protein fusion from the pFastbac vector described above and added a cassette containing the attR recombination sites flanking a ccdB gene plus a chloramphenicol-resistance gene. The baculovirus vector was propagated using ccdB Survival 2 competent cells (available from Thermo Fisher Scientific, Waltham MA USA, Cat # A10460).
  • This final baculovirus vector is the parent vector for the NMDAR and Kir2.1 BacMam expression vectors used in these baculovirus vectors.
  • BacMam virus production in E. coli and insect cells For the production of recombinant baculovirus vectors (bacmids), we followed the directions in the Invitrogen pFastBac manual. See, Thermo Fisher Scientific, Waltham MA USA. In this system, recombination occurs in a permissive E. coli strain, DHI OBacTM, between two Tn7 recombination sites in the expression plasmid and a Tn7 transposase mini-attTn7 element present in a bacmid in the DHI OBacTM E. coli cells. As described above, this bacmid is a baculovirus shuttle vector containing the majority of the baculovirus genome. After transformation of DHI OBacTM cells, recombinant bacmid are recognized by a blue- white color change.
  • Bacmid DNA is prepared from white colonies and is used to transform insect cells. Approximately 5-6 days after transformation, the insect cells lyse due to their infection with baculoviruses. The cell supernatants which contain the P1 viruses were filtered and added to 150 ml of Sf9 (Spodoptera frugiperda) insect cells at 2x10 6 /ml in shaker flasks, and incubated in an INFORS shaking incubator at 27°C at 100 rpm for 5-6 days to generate P2 viruses. P3 viruses were generated using P2 viruses using a 1 :100 (v/v) dilution of the virus-containing supernatant to insect cells at 2x10 6 /ml.
  • NMDAR expression levels correlated with the amount of virus added, demonstrating the advantage of now making it possible neuroscientists and biochemists to titrate the expression level of NMDAR by adjusting the amount of virus.
  • baculovirus-expressed NMDAR protein was functional, as demonstrated by manual patch clamp measurements of NMDAR-dependent currents in the baculovirus- transduced HEK293 cells.
  • the glycine binding site antagonist MDL105.519 - but not ketamine or MK801 - protects cells from cytotoxicity and facilitates the measurement of NMDAR activity in a functional assay
  • Cytotoxicity is manageable with NMDAR inhibitors.
  • ketamine- mediated or MK801 -mediated protection of the cells from excitotoxicity would also enable us to measure NMDAR activity in a calcium flux assay.
  • HEK293 cells were transduced with two different amounts of baculovirus vectors encoding for NR1/2A and treated with channel blockers (ketamine or MK801) at the indicated concentrations. Sixteen hours after transduction, the cells were washed several times with assay buffer to remove the inhibitors and loaded with calcium 6 dye. NMDAR activity was measured on a fluorescent plate reader (FDSS) after addition of 100 ⁇ glycine and glutamate. Cell viability was assesses by CellTiter-Glo ® after the measurement.
  • FDSS fluorescent plate reader
  • Ketamine and MK801 are NMDAR channel blockers that prevented NMDAR- mediated excitotoxicity. We were not able to measure NMDAR-mediated calcium influx in the cells even after multiple washes of the cells. Thus, ketamine-mediated and MK801 - mediated protections from excitotoxicity are not suitable for the measurement of NMDAR activity in a microplate based assay, likely because of the inability to completely wash out the inhibitors. See, FIG. 2 "Protection with channel blocker (Current state of the art)".
  • HEK293 cells were transduced as described above and treated with the indicated inhibitors.
  • Cell viability was measured using CellTiter-Glo ® . The data were normalized to non-transduced cells in the presence of solvent and represents the mean ⁇ standard deviation (STDEV) of a representative experiment.
  • TCD201 allosteric negative modulator
  • MDL105,5 NMDAR ligand site binding antagonist
  • AP5 or MgCI 2 AP5 or MgCI 2
  • HEK293 cells were transduced with NMDAR-encoding baculovirus vectors as described above and loaded with calcium 6 dye. NMDAR-mediated calcium flux was measured after stimulation with 100 ⁇ glycine/glutamate. Data shown in TABLE 5 represent the mean ⁇ STDEV of the maximal fluorescence ratio (maximal fluorescence/baseline fluorescence, F max /F 0 )) of a representative experiment. Units are mM for MgCI 2 and ⁇ for all other inhibitors.
  • TABLE 6 shows the titration of the maximal NMDAR-mediated calcium flux in HEK293 cells transduced with baculovirus.
  • Cells were transduced with different ratios/amounts of NR1 and NR2A in the presence of MDL105.519 and NMDAR-mediated calcium flux was measured as in (B) after washout of MDL105,519.
  • a multiplicity of infection (MOI) ratio of 250xNR1 : 500xNR2A was found to be optimal and was used for all subsequent experiments.

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Abstract

La présente invention concerne un nouveau dosage à haut débit et des procédés d'étude des récepteurs de N-méthyl-D-aspartate (NMDAR). Le procédé permet une comparaison rapide de différentes sous-unités, la capacité de mesurer des effets des agonistes à la fois pour le site de liaison à la glycine et pour le site de liaison au glutamate 5. Les procédés exploitent l'utilisation de faibles sites de glycine et de glutamate se liant à des antagonistes plutôt qu'à la kétamine ou MK -801 pour atténuer la toxicité cellulaire et conserver le site de liaison au ligand dans un état sans ligand. Le procédé peut utiliser des vecteurs de baculovirus pour introduire des quantités titrables de différentes sous-unités de récepteurs. Le dosage reproduit la pharmacologie des NMDAR en réponse à des antagonistes et des modulateurs allostériques 10 connus, ainsi que la sensibilité au magnésium.
PCT/IB2016/057843 2015-12-22 2016-12-20 Méthode de dosage à haut débit pour identifier des modulateurs du récepteur de nmda allostériques WO2017109709A2 (fr)

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CN111982866A (zh) * 2020-06-05 2020-11-24 中国科学院新疆理化技术研究所 一种比色荧光猝灭双模检测氯胺酮的方法
CN114957445A (zh) * 2022-05-31 2022-08-30 陕西脉元生物科技有限公司 一种nmdar nr1亚基、nmdar的突变体及其构建方法和应用
WO2023021102A1 (fr) * 2021-08-20 2023-02-23 Bayer Aktiengesellschaft Utilisation de polypeptides ayant une activité d'indicateur de calcium pour identifier l'activité de protéines insecticides

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