Method for evaluating the binding affinity of odor molecules

Abstract

This invention provides a simple method for evaluating whether or not a peptide binds to isovaleric acid. [Solution] A method for evaluating the binding affinity to isovaleric acid, comprising a preparation step of preparing a fluorescently labeled compound in which a fluorescent group is bonded to the α-carbon atom of isovaleric acid, and a contact step of contacting the fluorescently labeled compound with a peptide fragment of an olfactory receptor.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the binding property of an odor substance, and particularly to a method for evaluating the binding property of isovaleric acid to a peptide.

Background Art

[0002] Olfaction is a sensory mechanism for detecting various odor substances represented by volatile organic compounds and the like, and is caused by the binding of an odor substance to an olfactory receptor. An olfactory receptor (OR: Olfactory Receptor) is present in olfactory nerve cells in the olfactory organ and is a G protein-coupled receptor having a seven-transmembrane structure. There are hundreds to thousands of types of olfactory receptors depending on the biological species, and they can bind to odor substances in various patterns to detect innumerable odors. Thus, since the relationship between olfactory receptors and odor substances is a many-to-many relationship, detailed research on olfactory receptors has been underway.

[0003] By the way, isovaleric acid is cited as a typical odor substance. Isovaleric acid is a pungent odor accompanied by unpleasant feelings such as body odor and foot odor, and is a cause of malodor pollution generated from livestock manure and the like. As human olfactory receptors that bind to this isovaleric acid, OR11H4, OR11H6, OR11H7 and OR51E1 are known (see Non-Patent Documents 1 to 3).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] Isovaleric acid binds to olfactory receptors such as OR11H4, but because olfactory receptors have long amino acid sequences, various other odorants also bind to these receptors. Therefore, if the entire olfactory receptor is used as the sensing part of an odor sensor, it can be difficult to distinguish between isovaleric acid and other odorants. Thus, it is desirable to identify peptide fragments consisting of amino acid sequences that bind to isovaleric acid within the amino acid sequence structure of the olfactory receptor. However, no simple method for evaluating such peptide fragments is currently known.

[0006] The present invention aims to provide a method for easily evaluating whether or not a peptide fragment binds to isovaleric acid. [Means for solving the problem]

[0007] A first aspect of the present invention is a method for evaluating the binding affinity to isovaleric acid, comprising a preparation step of preparing a fluorescently labeled compound in which a fluorescent group is bonded to the α-carbon atom of isovaleric acid, and a contact step of contacting the fluorescently labeled compound with a peptide fragment of an olfactory receptor. [Effects of the Invention]

[0008] According to the evaluation method of the first embodiment, the binding affinity of peptides to isovaleric acid can be easily evaluated. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows an example of the synthesis of the peptide fragment to be evaluated. [Modes for carrying out the invention]

[0010] 1. First Embodiment The first embodiment of the present invention is a method for evaluating the binding affinity of a peptide to isovaleric acid, comprising a preparation step, a contact step, and a measurement step in that order. These steps will be described below.

[0011] (Preparation steps) This step involves preparing a fluorescently labeled compound. The fluorescently labeled compound has a structure in which a fluorescent group is bonded to the α-carbon atom of isovaleric acid, and is a fluorescently labeled isovaleric acid analog. Specifically, it is represented by the following formula (1).

[0012] [ka]

[0013] R 1 R indicates a single bond or a divalent linking group. 2 This indicates a fluorescent group.

[0014] Examples of divalent linking groups include optionally substituted hydrocarbon groups, -NHC(S)NH-, -NHC(O)NH-, -NHCOO-, -NHCO-, -COO-, -CO-, -O-, and combinations thereof. Examples of hydrocarbon groups include alkylene groups having 1 to 10 carbon atoms, such as methylene, ethylene, n-propylene, isopropylene, n-butylene, and cyclohexylene; and arylene groups having 6 to 20 carbon atoms, such as phenylene, methylphenylene, and naphthylene. If the hydrocarbon group is substituted, examples of substituents include halogen atoms such as F and Cl, and hydroxyl groups. 1 Preferably, these include -NHC(S)NH-, -NHC(O)NH-, -NHCOO-, and -NHCO-, and more preferably, -NHC(S)NH- and -NHC(O)NH-.

[0015] The fluorescent group is a group derived from a fluorescent dye, and is a monovalent functional group (residue) obtained by removing one hydrogen atom or the like from the fluorescent dye. Examples of such fluorescent dyes include fluorescein, rhodamine, cyanine dyes, eosin, perylene, pyrene, and their derivatives. Preferably, fluorescein and its derivatives (hereinafter referred to as fluorescein-based dyes) are used. In other words, it is preferable that the fluorescent group is derived from a fluorescein-based dye.

[0016] Fluorescently labeled compounds can be synthesized, for example, by reacting valine with a fluorescent dye. Since valine has a main skeleton similar to isovaleric acid and possesses an amino group, resulting in excellent reactivity, a fluorescent group can be easily added to it. That is, a compound of formula (1) having the main skeleton of isovaleric acid and a fluorescent group can be easily obtained.

[0017] The fluorescent dye used in the above synthesis preferably has a group capable of binding to the NH2 of valine. Examples of such groups include an isothiocyanate group, an isocyanate group, an ester group, a carboxyl group, and the like. The main skeleton of the fluorescent dye is the same as the above-described fluorescent group. Specific examples of the fluorescent dye include fluorescein isothiocyanate (FITC), fluorescein succinimidyl ester, and the like.

[0018] Since the fluorescently labeled compound prepared in this preparation step has a fluorescent group directly or indirectly bonded to the α-position carbon atom of isovaleric acid, it has a fluorescent group while retaining the main structure of isovaleric acid (that is, a carboxylic acid structure having 5 carbon atoms with an isopropyl group). Therefore, regarding the binding property to the peptide fragment, it has the same binding property as isovaleric acid. Therefore, in the first embodiment, by using this fluorescently labeled compound, isovaleric acid bound to the peptide fragment can be confirmed by fluorescence.

[0019] (Contact step) In this step, the peptide fragment of the olfactory receptor is contacted with the fluorescently labeled compound.

[0020] The olfactory receptor serving as the group of the peptide fragment is a seven-transmembrane G protein-coupled receptor present in the olfactory nerve cells of the olfactory epithelium. From the viewpoint of high binding property to isovaleric acid, preferably, OR11H4, OR11H6, OR11H7, OR51E1, etc. can be mentioned. These can be used alone or in combination of two or more.

[0021] The number of amino acid residues in the olfactory receptor peptide fragment is, for example, 50 or less, preferably 30 or less, more preferably 20 or less, and even more preferably 15 or less, and also, for example, 8 or more, preferably 10 or more. By setting the number of amino acid residues to be below the above upper limit, only the peptide portion with high binding affinity to isovaleric acid can be accurately identified. Furthermore, since the peptide fragment can be synthesized easily and in a short time, this evaluation method can be carried out reliably and in a short time. On the other hand, by setting the number of amino acid residues to be above the above lower limit, peptide fragments that can bind to isovaleric acid more reliably can be identified.

[0022] Methods for synthesizing such peptide fragments include, for example, (1) a method of synthesizing peptide fragments by sequentially extending the chain length of amino acids one by one using a peptide synthesizer, and (2) a method of obtaining peptide fragments by cleaving the peptide bonds of olfactory receptors with a protease. From the viewpoint of reliably obtaining peptide fragments consisting of a desired amino acid sequence, method (1) is preferred.

[0023] Multiple peptide fragments will be prepared for evaluation. Many peptide fragments can be obtained from the amino acid sequence of a single olfactory receptor, but they should be prepared appropriately considering the number of residues in the peptide fragments and the number of samples. For example, by referring to the amino acid sequence structure of a single olfactory receptor, peptide fragments with the desired number of amino acid residues can be synthesized sequentially at the desired intervals. More specifically, if the amino acid at the N-terminus of a single olfactory receptor is set as the 1st amino acid, the number of amino acid residues to be synthesized is set as m, and the difference in the order of the N-terminus amino acid residues between adjacent peptide fragments is set as k, then peptides consisting of the 1st to mth amino acid sequences, peptides consisting of the (1+k)th to (m+k)th amino acid sequences, peptides consisting of the (1+2k)th to (m+2k)th amino acid sequences, ..., peptides consisting of the (1+n×k)th to (m+n×k)th amino acid sequences can be synthesized (see Figure 1). This allows for the acquisition of a uniform and complete evaluation sample population from fragments obtained from a single olfactory receptor, enabling more accurate evaluation.

[0024] The method for contacting peptide fragments with fluorescently labeled compounds is to have multiple peptide fragments each come into contact with the fluorescently labeled compound. For example, each peptide fragment may be placed or immobilized in predetermined regions on one or more substrates, and then the fluorescently labeled compound may be placed in those regions. The fluorescently labeled compound may be placed alone, or it may be placed in a liquid state added to a buffer solution or the like. Examples of substrates include filter paper and plastic substrates.

[0025] After contact, it is preferable to wash the area with a washing solution such as a buffer. This removes any residue of the fluorescently labeled compound that did not bind to the peptide fragment.

[0026] (Measurement process) In this process, the luminescence intensity of the peptide fragment is measured. Specifically, the luminescence intensity of a fluorescently labeled peptide, to which a fluorescently labeled compound is bound, is measured.

[0027] Specifically, each fluorescently labeled peptide is irradiated with excitation light from a fluorescently labeled compound, and thus a fluorescent dye, and the emission is detected to obtain the emission intensity. The excitation light wavelength and emission wavelength are appropriately determined according to the fluorescently labeled compound. For detecting the emission, a fluorescence image analyzer, spectrophotometer, etc., can be used.

[0028] The level of emission intensity is then used to determine the isovaleric acid binding affinity of the peptide fragment. Specifically, for fluorescently labeled peptides with high emission intensity, the corresponding peptide fragment is judged to have high isovaleric acid binding affinity, and for fluorescently labeled peptides with low emission intensity, the corresponding peptide fragment is judged to have low isovaleric acid binding affinity.

[0029] Furthermore, when determining the level of emission intensity, for example, in addition to the above emission intensity (Intensity A), a reference emission intensity (Intensity B) using a reference fluorescently labeled compound may be used. Specifically, (1) a reference fluorescently labeled compound is prepared by attaching a fluorescent group to isovaleric acid or glycine similar to valine, (2) the reference fluorescently labeled compound is brought into contact with the peptide fragment, and (3) the emission intensity of the peptide fragment is measured to obtain the reference emission intensity (Intensity B). Then, the ratio of the emission intensity of the target substance (Intensity A) to the reference emission intensity (Intensity B) (Intensity A / Intensity B) is calculated, and the binding affinity can be determined based on the level of this ratio. This allows for a clearer evaluation of the binding affinity to isovaleric acid alone.

[0030] According to the evaluation method of the first embodiment, the binding affinity of isovaleric acid in peptide fragments can be easily evaluated. Consequently, peptide fragments with high isovaleric acid binding affinity can be easily identified. In particular, a large number of peptide fragments can be screened quickly and concisely. In conventional evaluation methods, isovaleric acid itself is brought into contact with and bound to the peptide fragment, then the bound isovaleric acid is desorbed or eluted, and then the desorbed isovaleric acid is measured by gas chromatography or the like. However, this conventional method requires both binding and desorbing of isovaleric acid, and the desorbed isovaleric acid must be measured by gas chromatography for each peptide fragment sample, which is time-consuming and laborious. In contrast, in the first embodiment, for example, various peptide fragments can be placed on a substrate, and their fluorescence intensities can be measured and evaluated simultaneously. Therefore, screening can be performed quickly and concisely.

[0031] Peptide fragments evaluated as having high binding affinity to isovaleric acid using this evaluation method can be suitably used, for example, in compositions for measuring isovaleric acid or in the sensing part of an odor sensor. Examples of odor sensors include field-effect transistor sensors, semiconductor sensors, quartz crystal oscillator sensors, and film-type surface-applied force sensors, with field-effect transistor sensors being preferred. Examples of field-effect transistor sensors include graphene field-effect transistors (GFETs) and molybdenum disulfide field-effect transistors.

[0032] 2. Appearance Those skilled in the art will understand that the exemplary embodiments described above are specific examples of the following embodiments.

[0033] (Section 1) An evaluation method according to one embodiment is a method for evaluating the binding affinity to isovaleric acid, which may comprise a preparation step of preparing a fluorescently labeled compound in which a fluorescent group is bonded to the α-carbon atom of isovaleric acid, and a contact step of contacting the fluorescently labeled compound with a peptide fragment of an olfactory receptor.

[0034] (Section 2) In the evaluation method described in Section 1, the fluorescently labeled compound may be synthesized in the preparation step by reacting valine with a fluorescent dye.

[0035] (3) The evaluation method described in paragraph 1 or 2 may further include a measurement step of measuring the luminescence intensity in the peptide fragment after the contact step.

[0036] (Article 4) In the evaluation method described in any one of paragraphs 1 to 3, the olfactory receptor may be at least one selected from the group consisting of OR11H4, OR11H6, OR11H7, and OR51E1.

[0037] (Clause 5) In the evaluation method described in any one of paragraphs 1 to 4, the fluorescent group may be derived from a fluorescein-based dye. [Examples]

[0038] The present invention will now be described in detail with reference to examples and reference examples, but the scope of the present invention is not limited thereto.

[0039] <Example 1> 1. Measurement of fluorescein-labeled valine (Preparation steps) Valine and fluorescein 5-isocyanate (isomer 1) were dissolved in 10% acetonitrile carbonate-bicarbonate buffer (pH 9.25), and the solution was stirred at room temperature for 24 hours, after which it was freeze-dried. The resulting powder was purified using a high-performance liquid chromatograph ("LC-20AR", Shimadzu Corporation). A TSKgel column (20 mm I.D., 25 cm, 5 μm, "ODS-80TS", Tosoh Corporation) was used for the chromatography column. This yielded fluorescein-labeled valine represented by the following formula (2). The obtained powder was confirmed to be fluorescein-labeled valine using a mass spectrometer (MALDI-TOFMS: "AXIMA Performance", Shimadzu Corporation).

[0040] [ka]

[0041] (contact process) Four types of olfactory receptors were selected: OR11H4, OR11H6, OR11H7, and OR51E1. A total of 223 peptide fragments of these olfactory receptors were synthesized directly onto filter paper using a fully automated microwave peptide synthesizer (for high-speed synthesis, manufactured by Biotage AB Initiator). Specifically, starting from the N-terminus of each olfactory receptor, peptide groups consisting of amino acid sequences from 4n+1 to 4n+12 were synthesized sequentially, starting with the amino acid sequence from the 1st to the 12th amino acid sequence, then from the 5th to the 16th amino acid sequence, and so on.

[0042] The fluorescein-labeled valine was added dropwise to each peptide region (223 locations) on the filter paper on which these peptides were synthesized, bringing each peptide fragment into contact with the fluorescein-labeled valine. The filter paper was then washed with phosphate-buffered saline.

[0043] (Measurement process) Next, using a fluorescence scanner ("Tyhoon FLA 9500," manufactured by GE Healthcare Japan), light at a wavelength of 494 nm was irradiated onto each peptide region (223 locations) on the filter paper, and the fluorescence intensity (Intensity A) at an emission wavelength of 520 nm was measured.

[0044] 2. Measurement of fluorescein-labeled glycine (reference fluorescently labeled compound) The procedure was carried out in the same manner as above, except that glycine was used instead of valine. The fluorescence intensity (Intensity B) at an emission wavelength of 520 nm was then measured for each peptide region (223 locations) that was contacted with fluorescein-labeled glycine, as shown in formula (3) below.

[0045] [ka]

[0046] 3. Evaluation For each peptide region (223 locations), the fluorescence intensity ratio (Intensity A / Intensity B) of fluorescein-labeled valine to fluorescein-labeled glycine was calculated. The average value (Av) of the fluorescence intensity ratio for these 223 peptide fragments was 0.950, and the standard deviation (SD) was 0.128. The value obtained by adding twice the standard deviation to the average value (Av + 2 × SD = approximately 1.2) was used as the baseline value for statistical usefulness. The peptide fragments that showed high fluorescence intensity ratios exceeding this baseline value were the peptide fragments with sequence numbers 1 to 13, shown in Table 1 below. Therefore, these 13 peptide fragments were judged to have high binding affinity to isovaleric acid. Similarly, when evaluated using the robust Z score, the values ​​for peptide fragments with sequence numbers 1 to 13 were also superior.

[0047] [Table 1]

[0048] <Reference example> The peptide fragment of Sequence ID No. 1 was synthesized on a piece of filter paper using a peptide synthesizer ("MultiPep RSi," Intavis AG). This peptide-coated filter paper was placed in phosphate-buffered saline containing 10 μM isovaleric acid and left at room temperature for 2 hours to allow isovaleric acid to bind to the peptide fragment. Subsequently, the peptide-coated filter paper was removed, washed with phosphate-buffered saline, and then placed in acetone solution. This allowed the isovaleric acid bound to the peptide fragment to be eluted into the acetone solution.

[0049] Next, the content of eluted isovaleric acid was measured using a gas chromatograph-mass spectrometer ("GCMS-TQ8050NX," manufactured by Shimadzu Corporation). The isovaleric acid content was determined based on a calibration curve that was prepared in advance using isovaleric acid standard solutions of known concentration.

[0050] The peptide fragments of SEQ ID NOs. 2-13, and the peptide fragments of SEQ ID NOs. 14-16 shown in the table below, were also measured for the amount of isovaleric acid that was bound to and eluted by isovaleric acid in the same manner as described above. These results are shown in Table 2 below.

[0051] [Table 2]

[0052] <Consideration> As is clear from the table, in the reference example, peptides SEQ ID NOs. 1-13 were found to have a high isovaleric acid content and high binding affinity to isovaleric acid. On the other hand, the presence of isovaleric acid was hardly detected in the other peptides, specifically peptides SEQ ID NOs. 14-16. This result was consistent with the evaluation method used in Example 1. Therefore, it can be concluded that the evaluation method of the present invention is effective.

Claims

1. A method for evaluating the binding affinity to isovaleric acid, A preparation step for a fluorescently labeled compound in which a fluorescent group is bonded to the α-carbon atom of isovaleric acid, and Contact step: Contacting the fluorescently labeled compound with a peptide fragment of an olfactory receptor. An evaluation method comprising the following features.

2. The evaluation method according to claim 1, wherein the preparation step involves synthesizing the fluorescently labeled compound by reacting valine with a fluorescent dye.

3. The evaluation method according to claim 1, further comprising a measurement step of measuring the luminescence intensity in the peptide fragment after the contact step.

4. The evaluation method according to claim 1, wherein the olfactory receptor is at least one selected from the group consisting of OR11H4, OR11H6, OR11H7, and OR51E1.

5. The evaluation method according to claim 1, wherein the fluorescent group is derived from a fluorescein-based dye.

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