Compositions for use in contacting receptor proteins

Abstract

To provide a technique for more efficiently performing a test for bringing a ligand and / or a ligand candidate substance into contact with a receptor protein.SOLUTION: A composition for use in contact with a receptor protein, comprising a plurality of types of ligands and / or ligand candidate substances.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compositions and the like for use in contacting receptor proteins. [Background technology]

[0002] In drug screening, screening using receptor proteins is performed. Specifically, a test compound is contacted with cells expressing the receptor protein that is the drug target, and the effectiveness of the test compound as a drug is evaluated based on the receptor protein responsiveness. Similar screening using receptor proteins is also performed to find new ligands for orphan receptor proteins.

[0003] The receptor protein-ligand relationship is like a key-lock relationship. For this reason, it is common to use one ligand for one receptor protein to evaluate affinity, efficacy, and concentration responsiveness. Multiple ligands may cause competitive inhibition at the same binding site on the receptor protein, or they may cause non-competitive inhibition through structural changes in the receptor protein due to binding to different binding sites on the receptor protein. Therefore, in screening using receptor proteins, each type of ligand / ligand candidate substance is brought into contact with the receptor protein. The number of types of ligand / ligand candidate substances is usually enormous, and there is a need to improve the efficiency of screening using receptor proteins. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 024902 Summary of the Invention [Problem to be solved by the invention]

[0005] Patent Document 1 discloses the use of cells expressing modified olfactory receptor proteins or lipid bilayer membranes containing modified olfactory receptor proteins as odor sensors. Artificially preparing lipid bilayer membranes containing olfactory receptor proteins is undesirable from the standpoint of manufacturing efficiency. Therefore, attention was focused on the use of cells expressing olfactory receptor proteins. Cells are complex systems, and their responsiveness changes from day to day or from measurement to measurement, making receptor protein quality testing using ligands necessary. For similar reasons as with receptor protein screening, it was thought that receptor protein quality testing would also require contacting each type of ligand with the receptor protein one by one. Given that cells expressing multiple olfactory receptor proteins are expected to be used as odor sensors, it is important to improve the efficiency of receptor protein quality testing using ligands.

[0006] An objective of the present disclosure is to provide a technique for more efficiently conducting tests in which a ligand and / or a ligand candidate substance is brought into contact with a receptor protein. [Means for solving the problem]

[0007] In light of the above-mentioned problems, the present inventors have conducted extensive research and found that contacting a receptor protein with multiple ligands results in less difference in the responsiveness of the receptor protein than contacting the receptor protein with only one ligand. Furthermore, the former case reduces the inter-test variability in measured values. Based on these findings, the present inventors have conducted further research and found that the above-mentioned problems can be solved by a composition containing multiple ligands and / or ligand candidate substances for use in contacting a receptor protein. Specifically, the present disclosure encompasses the following aspects.

[0008] Item 1. A composition for use in contacting a receptor protein, comprising multiple types of ligands and / or ligand candidate substances.

[0009] Item 2. The composition according to Item 1, wherein the total number of species of the ligands and the ligand candidate substances is 5 or more.

[0010] Item 3. The composition according to Item 1 or 2, which contains multiple types of the ligand.

[0011] Item 4. The composition according to Item 3, wherein the number of species of the ligand is 5 or more.

[0012] Item 5. The composition according to Item 3 or 4, wherein at least two or more of the plurality of ligands are ligands for different receptor proteins.

[0013] Item 6. The composition according to any one of Items 3 to 5, wherein at least two or more of the plurality of ligands are ligands for the same receptor protein.

[0014] Item 7. The composition according to any one of Items 1 to 6, wherein the total number of species of the ligands and the ligand candidate substances is 100 or less.

[0015] Item 8. The composition according to any one of Items 1 to 7, wherein the ligand and / or the ligand candidate substance is a volatile organic compound.

[0016] Item 9. The composition according to any one of Items 1 to 8, which does not contain a biological sample.

[0017] Item 10. The composition according to any one of Items 1 to 9, wherein the receptor protein is a receptor protein that is held in a lipid membrane.

[0018] Item 11. The composition according to any one of Items 1 to 10, wherein the receptor protein is an olfactory receptor protein.

[0019] Item 12. The composition according to any one of Items 1 to 11, which is used for testing the quality of a receptor protein or for screening for a ligand for a receptor protein.

[0020] Item 13. A method for measuring the responsiveness of a receptor protein, which comprises contacting a plurality of types of ligands and / or ligand candidate substances with the receptor protein. [Effects of the Invention]

[0021] The present disclosure provides a technique for more efficiently conducting a test in which a receptor protein is contacted with a ligand and / or a ligand candidate substance, and more specifically, a composition containing a ligand and / or a ligand candidate substance for use in the test. [Brief explanation of the drawings]

[0022] [Figure 1] The results of Test Example 1 are shown. The vertical axis shows the ratio of the luminescence value when a ligand-containing solution was added to the luminescence value when a ligand-free solution was added. The horizontal axis shows the insect olfactory receptor expressed by the cells used. In the legend, "Individual" indicates the case where a solution containing only one type of ligand for the insect olfactory receptor was added, and "Mixture" indicates the case where a solution containing a mixture of seven types of ligand was added. [Figure 2] The results of Test Example 2 are shown. "Individual" indicates the case where a solution containing only one type of ligand for an insect olfactory receptor was added, and "Mixture" indicates the case where a solution containing a mixture of 10 types of ligands was added. The vertical axis indicates the ratio of the luminescence value when a ligand-containing solution was added to the luminescence value when a ligand-free solution was added. The horizontal axis indicates the insect olfactory receptor expressed by the cells used. In the legend, "1st," "test," "2nd test," and "3rd test" each represent independent tests performed using different cells with different passage numbers. DETAILED DESCRIPTION OF THE INVENTION

[0023] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0024] In this specification, any combination of upper and lower limits of a certain numerical range is also directly and unambiguously disclosed.

[0025] In one aspect, the present disclosure relates to a composition (sometimes referred to herein as the "composition of the present disclosure") for use in contacting a receptor protein, which contains multiple types of ligands and / or ligand candidate substances. This is described below.

[0026] A ligand is a substance that can bind to a receptor protein and modulate (activate or inhibit) the signal transduction activity of the receptor protein and / or modulate the binding of the receptor protein to another ligand, and is not particularly limited thereto. Examples of ligands include low-molecular-weight compounds, fragrances, ions, peptides, proteins, sugar chains, nucleic acids, neurotransmitters, hormones, etc. Examples of low-molecular-weight compounds that serve as ligands include organic compounds with a molecular weight of 1,000 or less. In one embodiment, the molecular weight of a low-molecular-weight compound is 50 or more, 100 or more, or 150 or more, and can be 900 or less, 800 or less, 700 or less, 600 or less, or 500 or less. In one embodiment, a low-molecular-weight compound is a volatile organic compound. A volatile organic compound is a compound with a boiling point of, for example, 400°C or less, 350°C or less, 300°C or less, 250°C or less, 200°C or less, 150°C or less, 100°C or less, or 50°C or less.

[0027] Whether or not a substance is a ligand for a receptor protein can be determined as follows. If the activity signal derived from the receptor protein changes when the substance (test substance) is contacted with the receptor protein compared to when the test substance is not contacted, the test substance can be determined to be a ligand for the receptor protein. The activity signal used as an evaluation index in this determination may be a signal inherent to the cell (e.g., calcium ion amount, cAMP amount, etc.), or may be a signal obtained by converting such a signal (e.g., a signal derived from a substance that emits light in a calcium concentration-dependent manner).

[0028] In one embodiment, the ligand in the above-mentioned determination method is a substance that increases the activity signal derived from the receptor protein by, for example, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.5-fold or more, 3.0-fold or more, 5.0-fold or more, 6.0-fold or more, or 7.0-fold or more, compared to when the ligand is not contacted with the receptor protein, or a substance that increases the activity signal derived from the receptor protein by, for example, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.4-fold or less, 0.3-fold or less, or 0.2-fold or less.

[0029] A candidate ligand substance is a substance that has the potential to become a ligand for a receptor protein, although it has not yet been determined that the substance is a ligand. Its molecular species, molecular weight, etc. are the same as those of a ligand.

[0030] The total number of species of ligands and ligand candidate substances contained in the composition of the present invention is plural (i.e., two or more), and is not particularly limited as long as it is. From the viewpoint of the efficiency of the test in which the ligand and / or ligand candidate substance is contacted with the receptor protein, the total number of species is preferably three or more, four or more, five or more, six or more, seven or more, or eight or more. Taking into consideration the amount of solvent used in preparing the ligand, the total number of species is, for example, 100 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, or 15 or less.

[0031] In a preferred embodiment, the composition of the present invention contains multiple types of ligands. In this case, the composition of the present invention can be suitably used for quality testing of receptor proteins. From the viewpoint of efficiency of the test in which the ligands are contacted with the receptor protein, the number of types of ligands is preferably 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more. The total number of types is, for example, 100 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, or 15 or less, taking into account the amount of solvent used to prepare the ligand solution.

[0032] When the composition of the present invention contains multiple types of ligands, in one embodiment, at least two or more types of ligands can be ligands for different receptor proteins. From the viewpoint of efficiency of the test in which the ligands are contacted with the receptor protein, the number of types is preferably 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more. The total number of types is, for example, 100 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, or 15 or less, taking into consideration the amount of solvent used in preparing the ligand solution.

[0033] When the composition of the present invention contains multiple types of ligands, in one embodiment, at least two or more types of ligands may be ligands for the same receptor protein. The number of types is, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3.

[0034] When the composition of the present invention contains multiple types of ligands, in one embodiment, one type of ligand can be a ligand for two or more types of receptor proteins. The number of types is, for example, 2 to 15, 2 to 10, 2 to 5, 2 to 4, or 2 to 3.

[0035] The composition of the present invention may consist solely of a ligand and / or a ligand candidate substance, or may contain other components, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, and humectants.

[0036] The compositions of the present invention preferably contain as few substances as possible that may inhibit the interaction between the ligand and the receptor protein, other than the ligand and the ligand candidate substance. From this perspective, in one embodiment, the compositions of the present invention preferably do not contain biological samples (e.g., body fluids, skin, mucous membranes, internal tissues, etc., or purified samples thereof). From a similar perspective, when the compositions of the present invention contain low-molecular-weight compounds as the ligand and the ligand candidate substance, the protein concentration of the compositions of the present invention is preferably 10 mg / L or less, more preferably 1 mg / L or less, even more preferably 0.1 mg / L or less, even more preferably 0.01 mg / L or less, particularly preferably 0.001 mg / L or less, and particularly preferably 0 mg / L.

[0037] The receptor protein is not particularly limited as long as its signal transduction activity is regulated (activated or inhibited) by binding of a ligand. The receptor protein may be either a transmembrane receptor or an intracellular receptor (e.g., a cytoplasmic receptor, an intranuclear receptor), but is preferably a transmembrane receptor from the viewpoint of ease of testing in which a ligand and / or a ligand candidate substance is contacted with the receptor protein. When the receptor protein to be contacted with the composition of the present invention is a transmembrane receptor, the receptor protein is preferably held in a lipid membrane. The lipid membrane preferably has a composition closer to that of a cell membrane, for example, a lipid membrane containing phospholipids, more preferably a lipid membrane further containing glycolipids and / or sterols, and a cell membrane is particularly preferred. When the lipid membrane is a cell membrane, the receptor protein to be contacted with the composition of the present invention is preferably held in a cell.

[0038] The receptor protein is, for example, an ionotropic receptor and / or a G protein-coupled receptor. The receptor protein is preferably an olfactory receptor protein, and particularly preferably an insect olfactory receptor protein.

[0039] Insect olfactory receptor proteins are membrane proteins with seven transmembrane domains that function as odor sensors in living organisms. From the amino terminus (hereinafter sometimes referred to as the "N-terminus") to the carboxyl terminus (hereinafter sometimes referred to as the "C-terminus") of an olfactory receptor protein, they are composed of the N-terminal region (NT), the first transmembrane domain (TM1), the first extracellular loop (EC1), the second transmembrane domain (TM2), the first intracellular loop (IC1), the third transmembrane domain (TM3), the second extracellular loop (EC2), the fourth transmembrane domain (TM4), the second intracellular loop (IC2), the fifth transmembrane domain (TM5), the third extracellular loop (EC3), the sixth transmembrane domain (TM6), the third intracellular loop (IC3), the seventh transmembrane domain (TM7), and the C-terminal region (CT). In the present disclosure, each region is determined by structure prediction (default conditions) using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html).

[0040] When the receptor protein is an insect olfactory receptor protein, whether a substance is a ligand of a certain insect olfactory receptor protein can be determined, for example, as follows: A test substance is contacted with cells expressing a protein that emits fluorescence or luminescence in response to ions (such as calcium ions) that flow into the cells when (c) the insect olfactory receptor complex formed by (a) an insect olfactory receptor and (b) an insect olfactory receptor co-receptor responds, and the amount of luminescence from the cells is measured. If the amount of luminescence measured when the test substance is contacted changes compared to when the test substance is not contacted, the test substance can be determined to be a ligand of the receptor protein. Specifically, the measurement can be performed according to the method described in Patent Document 1.

[0041] The cells are not particularly limited, but animal cells such as insect cells and mammalian cells are preferred from the viewpoint of suitability for detecting the signaling activity of receptor proteins.

[0042] The cells preferably contain an exogenous polynucleotide containing a coding sequence for a receptor protein. This allows for the expression of any receptor protein and increases the expression level of the target receptor protein, thereby enhancing the detection sensitivity of the target ligand. The exogenous polynucleotide is a polynucleotide containing a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of the cell, and is not particularly limited thereto.

[0043] The composition of the present invention is used to contact a receptor protein. Specifically, for example, the composition of the present invention can be added to a compartment containing the receptor protein.

[0044] The compartment is not particularly limited as long as it contains cells and an apparatus capable of holding the cells, and may be, for example, a well plate or a dish or other apparatus for holding cells.

[0045] The number of compartments containing receptor proteins is preferably multiple (i.e., two or more). The number is preferably 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. The total number of species is, for example, 300 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 15 or less.

[0046] The responsiveness of a receptor protein can be measured by contacting the composition of the present invention with the receptor protein. Use of the composition of the present invention enables such measurements to be performed more efficiently. For example, by using a composition of the present invention containing ligands for each of the multiple receptor proteins, it is not necessary to change the test solution containing the ligand for each receptor protein. Furthermore, when screening for ligands for receptor proteins, using a composition of the present invention containing multiple candidate ligand substances can more efficiently determine whether the target ligand is present in the library compared to using each candidate ligand separately. As another example, use of a composition of the present invention containing multiple candidate ligand substances can reduce variability in measured values ​​between multiple measurements, thereby enabling more accurate measurements and evaluations while reducing the number of measurements.

[0047] For this reason, the composition of the present invention is particularly suitable for receptor protein quality testing or screening for ligands for receptor proteins. When used for receptor protein quality testing, for example, by adding the same composition of the present invention to each compartment of a cell chip containing multiple compartments, each compartment containing different receptor proteins (preferably cells in which the receptor protein is exogenously expressed), quality testing of multiple receptor proteins can be efficiently performed. Furthermore, when used for ligand screening, for example, by adding the composition of the present invention to each compartment of a cell chip containing a receptor protein (preferably cells in which the receptor protein is exogenously expressed), ligands that respond to the receptor protein can be efficiently screened.

[0048] In one aspect, the present invention relates to a method for measuring the responsiveness of a receptor protein, which comprises contacting a receptor protein with multiple types of ligands and / or ligand candidate substances. In this aspect, the contacting of the receptor protein with the multiple types of ligands and / or ligand candidate substances may be simultaneous (e.g., by using the composition of the present invention) or sequential, for example, by adding the multiple types of ligands and / or ligand candidate substances one by one to a compartment containing the receptor protein. [Example]

[0049] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0050] Test Example 1. Ligand Mixture Test 1 The seven ligands used in this study were previously confirmed to act as ligands for one, two, or three of the 14 insect olfactory receptors (OR_A to OR_N) also used in this study.

[0051] For each of the 14 insect olfactory receptors, we introduced the insect olfactory receptor co-receptor (Orco) gene and the GFP-aequorin gene into SpIm cells (cells derived from the mulberry butterfly, Spilosoma imparilis), and established stable expressing cells by selection using a selectable marker.

[0052] In these stably expressing cells, when the olfactory receptor complex responds to a ligand, calcium ions flow into the cell, and the GFP-aequorin protein expressed in the cytoplasm emits light in a calcium ion concentration-dependent manner. In other words, receptor responsiveness can be evaluated by measuring the luminescence intensity of the stably expressing cells.

[0053] To each of the 14 stably expressing cells expressing one of the 14 insect olfactory receptors, a solution containing one of the ligands that responds to the expressed insect olfactory receptor or a ligand-free solution was added, and the luminescence value was measured. The ratio of the luminescence value when the ligand-containing solution was added to the luminescence value when the ligand-free solution was added was calculated (Test a: Individual in Figure 1). Next, to each of the 14 stably expressing cells, a solution containing a mixture of seven ligands (the concentration of each ligand was the same as in Test a) or a ligand-free solution was added, and the luminescence value was measured. The ratio of the luminescence value when the ligand-containing solution was added to the luminescence value when the ligand-free solution was added was calculated (Test b: Mixture in Figure 1).

[0054] The specific procedures for tests a and b are as follows: 3.5 × 10 stably expressing cells were established. 4 Cells were seeded at 1000 cells / well in a 384-well plate and cultured at 27°C under atmospheric conditions for 24 hours. The medium was then replaced with 40 μL of Sf-900III medium containing a luminescent substrate and cultured for another 24 hours under atmospheric conditions. Ligand addition and luminescence measurement were performed using an FDSS / μCELL (Hamamatsu Photonics). Ligands were prepared fresh from the same DMSO stock. A baseline measurement was performed for 30 seconds, and 30 seconds after the start of the measurement, 10 μL of the ligand or Hanks'-HEPES buffer containing DMSO (the ligand solvent) was added. Luminescence values ​​were then measured every 2 seconds for 120 seconds, and the integrated luminescence values ​​at each time were used as the measured value.

[0055] The results are shown in Figure 1. Comparing the addition of only one ligand (Individual) with the addition of a ligand mixture (Mixture), the responsiveness of Mixture was slightly enhanced for OR_A, C, D, and G, but no difference in responsiveness was observed between Mixture and Individual for other receptors.

[0056] Test Example 2. Ligand Mixture Test 2 The 10 ligands used in this test were confirmed in advance to act as ligands for one, two, or three of the 14 insect olfactory receptors (OR_A to OR_N) also used in this test. Tests corresponding to tests a and b in Test Example 1 were performed three times each, and the differences in the measured values ​​between tests were examined.

[0057] The specific procedure is as follows: 14 types of stably expressing cells (Test Example 1) were prepared in Sf-900III medium containing a luminescent substrate, and 3.5 × 10 4 Two 384-well plates were seeded with 50 μL of cells / well and then cultured for 20 hours. Ligands were prepared fresh from the same DMSO stock. Each ligand was added individually to one plate, and a mixture of the ligands was added to the other. Ligand addition and luminescence measurement were performed using an FDSS / μCELL (Hamamatsu Photonics). A baseline measurement was performed for 30 seconds, and 30 seconds after the start of measurement, 12.5 μL of the ligand or Hanks'-HEPES buffer solution containing DMSO, the ligand solvent, was added. Luminescence values ​​were then measured every 2 seconds for 120 seconds, and the maximum luminescence value was used as the measured value. The test was performed in 1 st, , 2 nd , 3 rd Do this three times in total, st and 2 nd For two weeks, nd and 3 rd The experiment was carried out using cells that had been subcultured for an additional week.

[0058] The results are shown in Figure 2. Comparing the addition of only one ligand (Individual) with the addition of a mixture of ligands (Mixture), the inter-test difference in the measured values ​​for OR_A was greater with Mixture, but for many other receptors, the inter-test difference tended to be reduced with Mixture.

Claims

1. A composition for use in contacting a receptor protein, comprising multiple types of ligands and / or ligand candidate substances.

2. The composition according to claim 1 , wherein the total number of species of the ligands and the ligand candidate substances is 5 or more.

3. The composition of claim 1 , comprising multiple types of said ligands.

4. The composition of claim 3, wherein the number of species of the ligand is 5 or more.

5. The composition according to claim 3, wherein at least two or more of the plurality of ligands are ligands for different receptor proteins.

6. The composition according to claim 3, wherein at least two or more of the plurality of ligands are ligands for the same receptor protein.

7. The composition according to claim 1 , wherein the total number of species of the ligands and the ligand candidate substances is 100 or less.

8. The composition of claim 1 , wherein the ligand and / or the ligand candidate is a volatile organic compound.

9. The composition according to any one of claims 1 to 8, which does not contain a biological sample.

10. The composition according to any one of claims 1 to 8, wherein the receptor protein is a receptor protein that is held in a lipid membrane.

11. The composition according to any one of claims 1 to 8, wherein the receptor protein is an olfactory receptor protein.

12. The composition according to any one of claims 1 to 8, which is used for testing the quality of a receptor protein or for screening for a ligand for a receptor protein.

13. A method for measuring the responsiveness of a receptor protein, which comprises contacting a plurality of types of ligands and / or ligand candidate substances with the receptor protein.

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